Regulation of the function of mast cells and basophil granulocytes

by complement C3a-derived peptides;

in vitro and in vivo studies

Hajna Péterfy

PhD School of Biology

Head: Prof. Anna Erdei, DSc

Programme of Immunology

Head: Prof. Anna Erdei, DSc

Supervisor: Prof. Anna Erdei, DSc

Department of Immunology, Institute of Biology

Eötvös Loránd University, Budapest, Hungary

Table of contents

List of abbreviations................................................................................................................... 4

1.Introduction ............................................................................................................................. 6

1.1.Mast cells.............................................................................................................. 6 1.1.1. Origin, development and heterogeneity of mast cells ............................... 6 1.1.2. Basophil granulocytes ............................................................................... 8 1.1.3. The role of MCs in the immune system .................................................... 9 1.1.4. The role of MCs in disease...................................................................... 11

1.2. Allergy............................................................................................................... 12 1.2.1. The pathomechanism of allergic disease................................................. 12 1.2.2. Therapy.................................................................................................... 13

1.3. High affinity IgE receptor (FcεRI) .................................................................... 16 1.3.1. Structure .................................................................................................. 16 1.3.2. FcεRI-coupling signaling network .......................................................... 18

1.4. The complement system.................................................................................... 21 1.4.1. Activation and biological functions of the complement system ............. 21 1.4.2. The complement system and MCs .......................................................... 24

2.Aims ...................................................................................................................................... 26

3.Materials and Methods .......................................................................................................... 27

3.1. Materials............................................................................................................ 27 3.1.1. Buffers, media ......................................................................................... 27 3.1.2. Antibodies ............................................................................................... 31 3.1.3. Reagents .................................................................................................. 31

3.2. Methods ............................................................................................................. 33 3.2.1. Cells......................................................................................................... 33 3.2.2. Differentiation of mast cells from bone marrow derived precursors ...... 33 3.2.3. Degranulation assessed by the β-hexosaminidase assay......................... 34 3.2.4. Immunoprecipitation and Western blotting............................................. 35 3.2.5. Laser Scanning Confocal Microscopy .................................................... 35 3.2.6. Internalization assay ................................................................................ 36 3.2.7. Flow cytometric detection of FcεRI-β-chain .......................................... 36 3.2.8. Monitoring free cytosolic Ca2+ ion concentration................................... 36 3.2.9. Cytokine secretion assay ......................................................................... 37 3.2.10. Passive Systemic Anaphylaxis (PSA) and measurment of histamine in the serum of treated mice ..................................................................................... 37 3.2.11. Basophil activation test ......................................................................... 37 3.2.12. Statistical analyses................................................................................ 39

4.Results ................................................................................................................................... 40

4.1. The complement-derived inhibitory peptide C3a9 colocalizes with antigen-clustered FcεRI on MCs ........................................................................................... 40 4.2. C3a9 binds to the extracellular domain of FcεRI-β-chain ................................ 41 4.3. Treatment of MCs with C3a9 enhances the internalization of FcεRI after receptor clustering .................................................................................................... 42 4.4. Binding of C3a9 to MCs causes dissociation of the FcεRI-β-subunit bound PTK Lyn and Fyn.............................................................................................................. 44

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4.5. C3a9 inhibits the phosphorylation of several intracellular proteins including Lyn, the β subunit of FcεRI and PI3K...................................................................... 45 4.6. The effect of C3a9 on the elevation of [Ca2+]i .................................................. 46 4.7. C3a9 inhibits the MAPK-pathway .................................................................... 47 4.8. C3a9 inhibits secretion of the inflammatory cytokines..................................... 49 4.9. F-actin does not play a role in the C3a9 induced inhibition.............................. 50 4.10. Inhibition of Passive Systemic Anaphylaxis (PSA) in mice pretreated with the complement-derived peptide .................................................................................... 52 4.11. Testing newly designed peptides and peptidomimetics on the degranulation of RBL-2H3 .................................................................................................................. 53 4.12. The effect of monomeric and dimeric forms of C3a9-derived peptides in Passive Systemic Anaphylaxis (PSA) ...................................................................... 54 4.13. The effect of monomeric and dimeric forms of C3a9-derived peptides on the activation of human basophil granulocytes .............................................................. 55 4.14. The effect of commercially available anti-allergic drugs on the activation of human basophil granulocytes; comparison with peptide C3a9 ................................ 57

5. Discussion ............................................................................................................................ 60

Reference list ............................................................................................................................ 69

Summary .................................................................................................................................. 80

Összefoglalás............................................................................................................................ 81

Acknowledgements .................................................................................................................. 82

List of publications................................................................................................................... 83

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List of abbreviations

APC antigen presenting cell

BMMC Bone marrow-derived mast cell

Btk Bruton’s tyrosine kinase

C3 complement component 3

CCL CC - chemokine ligand

CXCL CXC - chemokine ligand

c-kit (CD117) receptor of stem cell factor

CR complement receptor

DAG diacylglycerol

DC dendritic cell

DNP dinitrophenyl

EAE Experimental Autoimmune Enchephalitis

ER endoplasmic reticulum

ERK Extracellular Signal-Regulated kinase

FcεRI high affinity IgE receptor

FITC fluorescein isothiocyanate

Gab2 Grb2-associated binding protein 2

GM-CSF granulocyte-macrophage colony stimulation factor

IFN interferon

Ig immunoglobulin

IL interleukin

IP3 inositol-triphosphate

ITAM immunoreceptor tyrosine –based activation motif

JNK c-Jun amino-terminal kinase

LAT linker for activation of T cell

LT leukotriene

MAC membrane-attack complex

MAPK mitogen-activated protein kinase

MC mast cell

MHC Major Histocompability Complex

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NFκB nuclear factor-κB

NTAL non-T cell activation linker

p38 MAPK of 38 kDa

PE phycoerythrine

PAF platelet activating factor

PG prostaglandin

PH pleckstrin homology domain

PI phosphatidilinositol

PI(3,4)P2 phosphatidilinositol-3,4-biphosphate

PI(4,5)P2 phosphatidilinositol-4,5-biphosphate

PI(3,4,5)P3 phosphatidilinositol-3,4,5-triphosphate

PI3K phosphoinositide 3-kinase

PKB (Akt) Protein kinase B

PKC Protein kinase C

PLCγ phospholipase Cγ

RBL-2H3 Rat Basophil Leukemia cell line

PTK protein tyrosine kinase

SCF stem cell factor

SH2 Src homology domain 2

TGF transforming growth factor

Th helper T cell

TLR Toll-like receptor

TNF tumor necrosis factor

TReg regulatory T cell

VEGF vascular endothelial growth factor

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1.Introduction

The basic role of the immune system is to defend the host against various bacteria, viruses

and parasites by recognition of pathogens and distinction self to non-self structures.

Sometimes this function of immune cells is defected and they recognize self structures as non-

self and induce an immune response against the host, generating autoimmune diseases. On the

other hand immune cells can recognize and respond to innocuous molecules in certain

circumstances, generating undesirable reactions, such as the allergic response. The main

effector cells of the allergic disorders are the mast cells. These cells are mostly activated

through their FcεRI, the high affiniy IgE-receptor. Upon IgE-mediated mast cell activation

several mediators are released which are responsible for the symptoms of allergic disorders.

Mast cells also express several receptors besides the high affinity IgE receptor, such as

complement receptors and TLRs, via which they can be activated in an IgE-independent-way.

As many allergens are able to activate the complement system, our group aimed to investigate

the relation between mast cells and the anaphylatoxic complement factors C3a and C5a,

which are released upon activation of the cascade.

1.1.Mast cells

1.1.1. Origin, development and heterogeneity of mast cells

Over a century ago, Paul Ehrlich proposed that mast cells (he used the term ‘mastzellen’,

i.e. well-fed cells, because their cytoplasm is stuffed with granules) may help to maintain the

nutrition of connective tissue. By now it is well accepted that these cells participate in the

regulation of both innate and adaptive immune responses (1,2). Mast cells (MCs) are resident

in tissues throughout the body but are the most common at sites that are exposed to the

external environment and often in proximity to blood and nerves, where they have a function

as sentinel cells in host defence (3).

These cells derive from CD13+CD34+CD117+ haematopoietic myeloid-cell progenitors

in the bone marrow (4,5). MC progenitors migrate to peripheral tissues, such as skin, mucosa

and airways, where they terminate their differentiation under the influence of factors in the

tissue microenvironment (6). Several cytokines, including IL-3, IL-4, stem cell factor (SCF),

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IL-9 and IL-10 regulate the maturation and phenotypes of mature MCs (7,8). Mitsui et al.

have described that the most relevant factor in MC development is SCF (9), and another group

has shown that c-kit, the receptor of SCF, and Gab2, which is included in the signalling

process of c-kit are essential in MC development and survival (10). Mature MCs are not a

homogenous population; they share many characteristics in different environment (8), they are

variant in cell size, cytoplasmic granule content and sensitivity to different stimuli (11).

There are two main types of MCs in rodents: the serosal type MCs residing in serosal

cavities, in the skin and the mucosal type MCs found mainly in the gastrointestinal mucosal

surfaces (12) (Table I.). In humans, two potentially analogous MC types, the so-called MCT

and MCTC, have been defined based on the protease content of their granules: they contain

either tryptase (MCT) or tryptase and also chymase (MCTC) (13). The large quantities of

tryptase and chymase that are synthesized by MCs suggest an important role of these

proteinases in the cells’ function. Tryptase seems to participate in proinflammatory function,

whereas chymase seems to be more involved in inflammatory reactions (8).

Table I. MC types in rodents

Cell-type Serosal type Mucosal type

Location serosal cavities, skin gastrointestinal mucosal surfaces

Life time years days

Plasma granules more less

Activation (besides FcεRI)

activated by peptidergic pathway non-activated by peptidergic pathway

C3aR és C5aR expressed non- expressed

Activation of MCs leads to the secretion of three classes of mediators: i., preformed

mediators stored in the cytoplasmic granules, such as vasoactives amines, neutral proteases,

cytokines and growth factors; ii., de novo synthesized lipid mediators, such as prostaglandins

and leukotriens; iii., cytokines, chemokines and growth factors (14) (Table II.).

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Table II. Main classes of mediators released by MCs

Mediators Examples of function

Granule-associated

Histamine and serotonin Enhance vascular permeability

Heparin Angiogenezis, enhances chemokine and cytokine function

Tryptase, chymase and other proteases Remodel tissue and recruit effector cells

TNF, VEGF Recruit effector cells and enhance angiogenezis

Lipid-derived

LTC4, LTB4, PGD2 and PGE2 Recruit effector cells, regulate immune response, promote angiogenezis

Platelet-activating factor Activates effector cells, enhances angiogenezis, induces inflammation

Cytokines

TNF-α, IL-1α, IL-1β, IL-6, IL-18, GM-CSF Induce inflammation

IL-3, IL-4, IL-5, IL-9, IL-13 Th2-type cytokines

IL-12 and IFN-γ Th1-type cytokines

IL-10, TGF-β and VEGF Regulate inflammation and angiogenezis

Chemokines

CCL2, CCL3, CCL4, CCL5, CCL11, CCL20 Recruit effector cells, inducing dendritic cells, regulate immune response

CXCL1, CXCL2, CXCL8, CXCL9, CXCL10 andCXCL11

Recruit effector cells and regulate immune response

Others

Nitric oxide Bactericidal

Antimicrobial peptides Bactericidal

1.1.2. Basophil granulocytes

MCs have been described as the tissue equivalent of basophil granulocytes, because both

cell-types contain basophilic plasma granules, release histamine and express FcεRI. However,

the development and the responsivness to various factors of these two cell-types are different

(15). Morphological and functional analyses indicate that human MCs are more closely

related to monocytes and macrophages, whereas basophil granulocytes share properties

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mainly with eosinophils (15,16) (Fig.1.). Basophils are circulating granulocytes that typically

mature in the bone marrow, circulate in the blood as mature cells, and can be recruited into

tissues at sites of immunological or inflammatory responses (17). Although basophil

granulocytes are different from human MCs in many aspects, they can be a good model for

the latter cells because the main activation route through FcεRI is common in both cell-types.

Figure 1.: Comparison between human MCs and other bone-marrow-derived cells.

Basophil granulocytes are similar to MCs in their FcεRI expression and histamine release.

MCs share the responsivness to IL-4 and bacterial products and nuclear morphology with

monocytes (15).

1.1.3. The role of MCs in the immune system

MCs are located at sites that interface the external environment, therefore they can play an

important role in the initiation and regulation of the immune response during infection. It is

well described that MCs are one of the main effector cells against nematodes and other

parasitic organism, and the proteases released upon the IgE-mediated activation are crucial in

this process (18). The role of these cells in the host’s response against bacterial, viral

pathogens has only recently begun widely accepted (2). MCs express several receptors which

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can recognize pathogens (e.g. TLRs) and this direct interaction is important in the initiation of

the early immune response. MCs can detect the opsonized pathogens also by Fc-receptors

(FcRs) and complement receptors (CRs) and in response to the different stimuli they can

release various types of mediators listed in Table II. (2). For example MCs activated by TLRs

release cytokines and chemokines but they do not degranulate in contrast to the FcR-mediated

activation (19-22) (Fig.2.). Therefore MCs are able to initiate the immune response to various

pathogens in different ways.

Figure 2.: Direct or indirect interactions between pathogens and MCs

A: Direct interactions include Toll-like receptor-mediated activation which does not lead

to degranulation, although they release cytokines, chemokines and lipid-mediators. B: Fc-

receptor-mediated activation leads to degranulation and also to the release of newly

synthesized mediators, resulting from either antigen-specific interactions with antibody or the

activations of B-cell superantigens. C: Mucosal type MCs can also be activated by receptors

for complement components C3a and C5a (2).

MCs also influence the initiation and duration of the adaptive immune response by various

products secreted during the innate immune response to pathogens (1). Migration, maturation

and function of dendritic cells (DCs), which are essential in the induction of adaptive immune

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response and T-cell activation, can also be regulated by the products of activated MCs. TNF

and IL-1 can facilitate the migration and functional maturation of DCs (23). Histamine can

enhance the expression of MHCII and costimulatory molecules on DCs and induces

maturation of DCs towards an effector DC2 phenotype, leading to the polarization of Th cells

to the Th2 direction (24,25). MCs are known to release several cytokines (Table II.) which

regulate T-cell dependent immune responses and induce T-cell polarization (26,27) and they

can enhance IgE production by released IL-4 and IL-13 (28). B cell development can also be

influenced by cytokines such as IL-6 and IL-13, secreted by certain MC populations (29-31).

Lu et al. had shown that MCs are essential in TReg-cell-dependent peripherial tolerance. In

this process activeted TReg-cells secrete IL-9, which is a growth and activation factor for MCs.

As a result, MCs release TNF-β leading to the induction of tolerance (32).

1.1.4. The role of MCs in disease

It is well known that the activation of MCs leads to allergic disorders such as asthma and

anaphylaxis (33,34). Allergic disease is described in details in the next chapter (1.2.).

These cells also have been shown to play a role in the early phase of autoimmune diseases

(35). It has been described that the number and distribution of MCs correlate with the

development of multiple sclerosis (36). Degranulation and an increased amount of proteolytic

enzymes - such as tryptase - in the cerebrospinal fluid suggest that activation of these cells

takes place during the pathogenesis of this disease (37,38). This reaction seems to be the

result of binding antibodies, inasmuch as in Experimental Autoimmune Enchephalitis (EAE),

the animal model of multiple sclerosis, the development of the disease was found to be

dependent on the expression of FcγRs by MCs (36). A similar reaction has been suggested in

the initiation of other autoimmmune disease, such as reumatoid arthitis (35).

MCs are known both to enhance and inhibit the growth of tumors. MC-derived heparin,

IL-8 and VEGF induce neovascularization, while histamine acts as an immunosuppressant.

Proteases released by activated MCs disrupt the surrounding matrix and facilitate metastases.

By contrast, TNF-α secreted by MCs induces apoptosis of tumor cells, tryptases activate

proteases-activated receptors and induce inflammation. An additional MC-mediator,

chondroitin sulfate inhibits tumor metastases. The beneficial and destructive mediators for the

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growth of tumors might be released from separate granules regulated by selectively distinct

signals and stimulations (39).

1.2. Allergy

1.2.1. The pathomechanism of allergic disease

The severity and incidence of allergic disorders is steadily increasing in the developed

countries. Allergic disease is a consequence of the exposure to normally innocuous substances

that elicit the activation of MCs through IgE-dependent or independent mechanisms (40),

therefore MCs take an important place in the investigastion of the mechanism of immediate

hypersensitive reactions. Allergic diseases are inflammatory disorders in which defective

immune regulation occurs; the allergen specific Th2 response is increased. Allergic

individuals usually have high levels of circulating IgE (41).

The physiological response to normally innocuous substances is thought to be a

consequence of adaptation of the immune system in the absence of endemic parasitic or

bacterial challenges. This „hygiene hypothesis” states that the defective development of

important immunological regulatory controls in early life results from inadequate exposure to

environmental microorganisms (42).

Allergens are taken up and processed by antigen presenting cells (APCs), such as dendritic

cells (DCs) and presented to T cells inducing a Th2 response. B cells produce allergen specific

IgE after isotype-switching caused by the Th2 cell derived lymphokines IL-4 and IL-13. IgE

then binds to the high affinity FcεRI expressed on the surface of MCs and basophil

granulocytes. Interaction of allergens with cell bound IgE causes MC activation, resulting in

the release of the contents of the granules, such as histamine, serotonin, cytokines and several

proteases eliciting the immediate hypersensivity reaction (40). MCs also produce cytokines

and lipid mediators, such as leukotriens and prostaglandins, which are the main contributors

to the late phase of allergic response (43). MC-derived IL-4 and IL-13 act on B cells while IL-

13 has also a role in hypersecretion of mucus and regulation of airway hypersensitivity. IL-5

is important for eosinophil infiltration and activation (41) (Fig.3.).

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Figure 3.: Allergic mechanism

Initial exposure to allergen leads to the activation of Th2 cells and IgE synthesis. IgE binds to

MCs and basophils, a process known as allergic sensitization. Subsequent exposure to

allergen causes IgE-sensitized MCs’ degranulation and release of preformed and newly

synthesized mediators. These include histamine, leukotrienes and cytokines, which promote

vascular permeability, smooth-muscle contraction and mucus production. This is the early

allergic response. In the late phase reaction chemokines and lipid mediators released by MCs

recruite Th2 cells and eosinophil granulocytes. Eosinophils release an array of pro-

inflammatory mediators and IL-3, IL-5 and IL-13 (41).

1.2.2. Therapy

In the Western world the severity and incidence of allergic disorders is increasing despite

the widespread use of steroids and other drugs. Therefore several investigations focus on the

development of novel therapies that prevent allergic disease (41,44) (Fig.4.).

One of the possible points of intervention is the inhibition of the development of allergen-

specific Th2 cells and prevention of IgE-production. The allergen-desensitization

immunotherapy promotes the allergen-specific immune response towards a Th1 response by

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injection of increasing doses of the allergen extract into the patients, however not all reports

agree on the increase in IFN-γ-associated response after treatment. The successful therapy is

associated with a decrease in the allergen-specific Th2 response and the induction of IL-10-

secreting allergen-specific TReg cells. The latter cells might inhibit the development of

allergen-specific immune response. During the desensitization immunotherapy the isotype of

the allergen-specific antibodies changes; there is an increase in the serum-levels of IgG1,

IgG4 and IgA instead of the production of IgE (41). In an other protocol where killed bacteria,

bacterial products or CpG vaccines are used, the allergen-specific immune response is shifted

towards Th1 or TReg. These effective vaccines inhibit the development of Th2 cells depending

on Th1 response and IFN-γ production. Another possible method of inducing strong and long-

lasting Th1 responses is by injections of DNA encoding a defined antigen. Transfected cells

produce antigen continuously which leads to a persistent long-lasting Th1 memory response

(44).

Another possibility is preventing IgE binding to FcεRI or inhibiting the activation of MCs

by co-engaging FcγRIIb inhibitory receptors with FcεRI. Humanized monoclonal antibodies

against the FcεRI-binding site of IgE have been used in humans with the aim of blocking MC

sensitization by IgE. Chimeric molecules that aimed at both targeting FcεRI-bearing cells and

exploiting the inhibitory properties of FcγRIIb have been described to inhibit IgE-induced

human MCs and basophil granulocytes activation (45).

A further possibility to inhibit allergic response is by blocking the function of the released

mediators. Treatment with antihistamines, leukotriene antagonists, neutralizing antibodies

specific for Th2 cytokines are widely applied (41).

Finally, the so-called „mast cell stabilizers” (e.g. chromolyn sodium) inhibit MCs from

releasing inflammatory mediators.

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Figure 4.: The potential points of intervention for treatment of allergy

Three main points of therapeutic intervention are proposed for the treatment of allergic

disease. a, (green box) block the initiation of immune response and development of allergen-

specific Th2 cells. b, (blue box) block the activation of allergen-specific Th2 cells, either

directly or indirectly through effects on the antigen-presenting cells. c, (grey box) block

effector molecules that cause the clinical symptoms of allergic disease (41).

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1.3. High affinity IgE receptor (FcεRI)

1.3.1. Structure

FcεRI is a multimeric cell-surface receptor that binds the Fc fragment of IgE with high

affinity. In humans it exists either as a tetrameric or as a trimeric complex (Fig.5.) and has a

wide tissue distribution (Table III). The tetrameric murine FcεRI is confined to MCs and

basophils (Table III).

Figure 5.: The structure of FcεRI

The tetrameric form of FcεRI (left figure) contains an α-chain, which is responsible for IgE-

binding, a β-chain and a dimer γ-chains, which contain ITAMs in their intracellular domain.

The trimeric form lacks the β-chain (right figure) (46).

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Table III. Comparison of human and murine FcεRI

Murine FcεRI Human FcεRI

Structure Tetrameric structure only Tetrameric and trimeric

structure

Expression pattern MCs and basophils MCs, basophils, monocytes

and macrophages, DCs,

eosinophils and platelets

Regulation of expression IL-4 does not enhance α-chain

expression

IL-4 enhances α-chain

expression

Binding properties Binds murine IgE Binds human and murine IgE

The tetrameric form contains an α-chain, a β-chain and a homodimer of disulphide-linked

γ-chains (47). The α-chain belongs to the immunoglobulin superfamily and comprises two

extracellular domains that bind a single IgE molecule, a transmembrane domain and a short

cytoplasmic tail that has no signaling function (46,47).

The FcεRI β- and γ-chains have no role in ligand binding (47). These chains each have an

immunoreceptor tyrosine-based activation motif (ITAM) which is tyrosine phosphorylated

after receptor crosslinking and leads to the association of the intracellular signaling molecules

by their Src homology 2 domains (SH2) (48,49). The β-chain is an amplifier of the FcεRI-

mediated signals (50,51), while the γ-chains are the main FcεRI-signaling units (46). These

latter two chains are shared with other Fc receptors, such as the high or the low affinity IgG

receptor or the IgA receptor (47).

Non-covalent association of the newly synthesized FcεRI-α-chain protein with the β-chain

and dimer γ-chains occurs co-translationally in the endoplasmic reticulum (ER). This

association is required to the poper folding, export from the ER and the cell surface

expression of the receptors (52). Murine FcεRI has an obligatory tetrameric form which

means that the β-chain is essential for the cell surface expression of FcεRI (53).

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1.3.2. FcεRI-coupling signaling network

IgE binding to the FcεRI might have more biological consequences than only sensitizing

the MCs (48). IgE binding alone can enhance the expression of FcεRI and induce MC survival

(54,55). The monomeric form of IgE has been divided into two groups: high cytokinergic and

poor cytokinergic IgEs. The high cytokinergic IgEs have been shown to elicit not only the

production and secretion of cytokines but also to initiate other activation events, such as

receptor internalization, degranulation and histamine synthesis (56,57). The FcεRI-β-chain is

known to have an amplifier role in the receptor coupling network (50) and its ITAM also

regulates the kinetics of gene transcription and the signaling pathway induced by monomeric

IgE (51).

IgE-mediated clustering of FcεRIs by antigen initiates a signaling pathway that results in

degranulation, i.e. the release of several preformed mediators, listed in Table II. Upon

prolonged stimulation a late-phase reaction is induced which leads to the secretion of lipid

mediators, chemokins and cytokines (47).

FcεRI cross-linking by antigen results in the translocation of the aggregated receptors into

lipid rafts (58). These membrane microdomains - which are rich in sphingolipids and

cholesterol and thought to control the organization of key coupling molecules, including

receptors, protein tyrosine kinase (PTKs) and adaptor molecules - have a crucial role in the

initiation of FcεRI-coupling network (59). The structural explanation for cross-linking

dependent FcεRI association with rafts is still not understood. Some studies showed that the

β-chain has an essential role in this process and pointed to the possible role of the

transmembrane segments of α- and γ-chain (60). Activated Lyn src kinases, which are

constitutively associated with FcεRI-β-chain (61), transphosphorylate the ITAMs on both the

β-chain and γ-chains and other Lyn kinases (62). Lyn has a higher specific activity in the

lipid-raft, although it can also be isolated from non-raft regions of the cell membrane..This

result suggests that the membrane microdomains somehow protect the activity of the Lyn

kinases (63). It is also described that the clustering of the receptor and the Lyn kinase

decreases the lateral mobility and this correlates with the activity of the Lyn kinase (64). It

had also been described that in this process actin has a structural or stabilizing role (65).

Fyn src kinase is also activated by binding to the ITAM of β-chain and is important for

phosphorylation of adaptor Gab2 (Grb2-associated binding protein 2) and activation of PI3K

(phosphoinositide 3-kinase), which leads to the degranulation of MCs (49,66,67). Fyn has

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also an important role in the activation of the MAPK (Mitogen Activated Protein Kinase)

pathway and nuclear translocation of NFκB is involved in the regulation and production of

several cytokines (68). PI3K catalyzes the production of PI(3,4)P2 and PI(3,4,5)P3 which can

bind several signaling molecules, such as Btk (Bruton’s tyrosine kinase), Akt, PLCγ2

(phospholipase Cγ), by their PH (Pleckstrin Homology) domain (48,49).

Syk, another protein kinase binds to the phosphorylated ITAM of the dimer γ-chains (69).

Lyn regulates the activation of Syk (70), however Lyn might not be the only PTK that

phosphorylates the ITAMs of FcεRI subunits and Syk (71). Lyn also phosphorylates adaptor

molecules, such as LAT (Linker for Activation of T cells) (72) and NTAL (Non-T cell

Activation Linker) (73). These proteins function as scaffolds and organize other signaling

proteins. PLCγ activation leads to the hydrolization of PI(4,5)PI2 into the second messengers

IP3 (inositol-triphosphate) and DAG (diacylglycerol). IP3 binds to the specific receptors in the

ER and triggers the release of Ca2+ from intracellular stores. The elevation of free cytosolic

Ca2+ concentration results in activation of the Ca2+-dependent PKC (Protein Kinase C), while

DAG activates the Ca2+-independent PKC (48). Activated PKC is essential for the IgE-

mediated MC degranulation (74) and directely activates several MAPKs and control cytokine

production (75,76). Tyrosine phosphorylation of enzymes and adaptors, including LAT,

NTAL, Vav, Grb2 and Sos stimulate small GTPase such as Ras directly or indirectly. These

pathways lead to the activation of the ERK, JNK and p38 MAP kinases and the

phosphorylation of transcription factors that induce the de novo synthesis and secretion of

cytokines (47,48) (Fig.6.).

The internalization of FcεRI after antigen-induced cross-linking is regulated by

ubiquitination which can act as a sorting signal to the endosomal/lysosomal pathway. FcεRI is

ubiquitinated in the raft microdomains upon receptor activation (77) and Cbl ubiquitin ligase

and also the scaffold protein CIN85 have a role in this process (78,79). Cbl and CIN85 not

only enhances receptor internalization but inhibits the activation of several signaling

molecules, cytokine production and degranulation upon IgE-mediated mast cell activation

(78-80). The ubiquitination of cross-linked FcεRIs by Cbl is dependent on the lipid raft

membrane region (77,80) and it can act as a dynamic signal triggering lateral removal of

proteins from rafts, thereby limiting the response (77).

It has been earlier described that actin has a crucial role in the initiation and sustainement

of raft polarization, and the distribution of proteins upon lymphocyte activation (81). In MC

activation actin has a similar role (82), but in addition it can also play a negative role in the

19

cells’ response initiated by FcεRI clustering (83). Actin inhibits both the early and the late

phase of MC activation by interfering with the association between FcεRI, Lyn kinase and

lipid rafts (84-86).

Figure 6.: Simplified scheme of FcεRI signaling events in MCs

Aggregation of FcεRI leads to translocation of receptors into lipid rafts, where the activated

Lyn src kinase phosphorylates ITAMs on the β- and γ-chains. This leads to Syk association

with the γ-chains which is followed by Syk activation. The phosphorylated ITAM of the β-

chain also binds the Fyn kinase. Lyn and Syk kinases phosphorylate several adaptor

molecules (e.g. LAT and NTAL) leading to the formation of the signaling complexes, which

are phosphorylated by Lyn and Syk. In parallel, Fyn src kinase phosphorylates Gab2, which

then associates with PI3K. Active PI3K produces PI(3,4)P2 and PI(3,4,5)P3 which bind

several signaling proteins, such as PLC-γ or Vav by their PH domain. Activated PLC-γ

catalyses the hydrolysis of PIP2 to IP3 and DAG, forming the „second messengers” which

activate PKC and induce the release of Ca2+ from internal stores. Both elevation of [Ca2+]i-

20

level and PKC activation induce degranulation. Activation of other enzymes and adaptors,

including Vav, Shc, Grb2 and Sos, stimulates small GTPases such as Ras, Rac and Cdc42.

These activation pathways lead to the activation of ERK, JNK and p38 MAP kinases,

phosphorylation of transcription factors and the expression and production of several

cytokines.

1.4. The complement system

1.4.1. Activation and biological functions of the complement system

The complement system is an integral part of both the innate and adaptive immune

systems and it is one of the major mechanisms to eliminate pathogens. Complement proteins

are present in the plasma and in various body-fluids, and they can be activated in a cascade-

like fashion. There are three pathways of activation (Fig.7.): i.) the classical pathway triggered

in most of the cases by antibody bound to the antigen; ii.) the mannose-binding lectin (MBL)

dependent pathway activated by mannose residues and other sugars present on the surface of

several pathogens; iii.) and the alternative pathway which is initiated by the deposited C3b

fragments, generated by the spontaneous hydrolysis of C3. All three pathways can be initiated

independently of pathogen specific antibodies as part of innate immunity. The early events in

each pathway consist of a sequence of cleavege reactions in which the larger component binds

covalently to the activator surface and participates in the activation of the next component of

the cascade. The three pathways converge in the activation of C3, the central component of

the complement system. C3b, the larger fragment of C3, bound covalently to the activator

surface is an integral component of the alternative pathway C3-convertase and the C5-

convertase enzymes, as well. C5b, generated by the cleavage of C5 initiates the assembly of

the membrane-attack complex (MAC) causing the lysis of various cells and pathogens

(87,88).

21

Figure 7.: Simplified scheme of the activation of the complement system.

The early events of all three pathways lead to the formation of the C3 convertase enzyme. This

is the point at which the three pathways converge and the cleveage of C3 results in the

generation of C3a and C3b. C3a, also called as anaphylatoxin, is a peptide mediator of

inflammation. Covalenty bound C3b opsonizes the pathogen. C5 convertase cleaves C5

generating C5a, which is another strong inflammatory peptide. Surface-bound C5b triggers

the late events in which the terminal components assemble to form the membrane-attack

complex (MAC) that can damage the membrane of pathogens or cells.

22

Activated components of the complement system can regulate and activate both the innate

and adaptive immune responses (Fig.8.). The central molecule of complement activation is

C3, which upon cleavage, generates a larger fragment, C3b, and the small inflammatory

peptide, C3a. Various types of complement receptors (CR) bind different activation fragments

of C3, such as C3a, C3b, iC3b and C3d. Complement receptors CR1, CR2, CR3, CR4 and

C3aR are expressed on the surface of several celly types, including phagocytes, lymphocytes,

MCs and erythrocytes. Fragments of the activated complement system enhance the

phagocytosis of the opsonized pathogens and activate cells through binding to these receptors.

C3a and C5a, the small cleavage fragment of C3 and C5 recruit inflammatory cells to the site

of infection and activate them by binding to their G-protein coupled receptors (87,88).

Figure 8.: Biological consequences of complement activation

Upon activation of the complement cascade several biologically active fragments are

generated, which exert their activity via interaction with their respective receptors expressed

on a wide variety of cell types show in the figure.

23

1.4.2. The complement system and MCs

As it has been detailed above the complement system plays an important role in several

immune processes – from the elimination of infectious agents to triggering and modulating

immune responses. MCs, which are also accepted players in these phenomena, express several

complement receptors, such as the C3a receptor (C3aR), C5a receptor (C5aR), CR2 and CR4

which bind C3d and iC3b, respectively (18,89). The role of MCs in the initiation of innate

immune response, particularly during bacterial infection, depends on these receptors and

therefore on complement activation, as well (90-92). It has been described that collectins and

the complement protein C1q can bind to the surface of several pathogens, thus activating the

cascade. Edelson et al. showed that α2β1 integrin can function as a receptor for C1q on MCs

and this receptor might function as a crucial co-receptor for the activation of these cells in

host defence that involves C1q (93). It has also been shown that the complement system can

induce and regulate DC migration and immune response trough the activation of MCs (94).

It is known that certain types of MCs express C3aR and C5aR while other types do not

bind the anaphylatoxic peptides, C3a and C5a (12) (Table I.). In MCs, which express C3aR

binding of C3a can act synergestically with the antigen-induced degranulation caused by

clustering of FcεRI (95).

Our group has previously described that the complement component C3a – but not C5a -

inhibits the FcεRI-mediated degranulation of the rat RBL-2H3 cell line, which has the

phenotype of mucosal-type MCs and express neither C3aR nor C5aR. It had been

demonstrated that the complement protein exerts its effect by binding to the extracellular loop

of the FcεRI-β-chain (96). We have identified a stretch of C3a, comprising residues 56-64,

that exert the inhibitory effect (97) (Fig.9.). Based on the 56-64 amino-acid sequence of C3a

two peptides were synthetized. Both the nonapeptide: (CCNYITELR) designated C3a7, and

its modified derivate the octapeptide C3a9 (DCCNYITR), were shown to inhibit the

degranulation of RBL-2H3 and BMMCs (Bone Marrow-derived Mast Cells). It has been

demonstrated that the complement-derived peptides also inhibit the IgE-mediated activation

of serosal type MCs, which can be activated by C3a (98). As it is known that for the

anaphylatoxic effect the 20 aminoacid long C-terminal end of C3a is needed, these data

clearly show that the sequence between positions 56-64 of C3a (C3a7) does not overlap with

the stretch of C3a which is important for binding to the C3aR on serosal type MCs.

24

Figure 9.: Schematic drawing of C3a structure

The C-terminal part of C3a is responsible for MC activation through C3aR. The 56-66

aminoacid sequence of C3a (red circle) inhibits the IgE-mediated activation of MCs. The

sequence of the two C3a-derived inhibitory peptides designated: C3a7, (a natural sequence),

and C3a9 (the modified derivative) is shown.

25

2.Aims

Our aims were to clarify the following:

● What is the molecular mechanism of the inhibition exerted by the C3a-derived

peptide?

● What is the effect of C3a9 on the late phase reaction of MCs?

● To investigate the peptides’ effect on human basophil granulocytes and in an in vivo

model.

● To develop more potent peptides and peptidomimetics based on the sequence of C3a9.

26

3.Materials and Methods

3.1. Materials

3.1.1. Buffers, media

-DMEM medium + 5% FCS

For RBL-2H3 cells

DMEM powder Dissolved in 1000ml distilled water

NaHCO3 3.7 mg/ml

Na-piruvate 0.22 mg/ml

L-glutamine 2 mM

Sreptomycin 0.1 mg/ml

Penicillin 100 U/ml

-RPMI medium 1640 + 10 % FCS

For Bone marrow-derived mast cells (BMMC)

RPMI powder Dissolved in 1000ml distilled water

NaHCO3 2 mg/ml

Na-piruvate 0.22 mg/ml

L-glutamine 2 mM

Sreptomycin 0.1 mg/ml

Penicillin 100 U/ml

27

-GKN

NaCl 8 g/l

KCl 0.4 g/l

NaH2PO4 1.77 g/l

Na2HPO4 0.77 g/l

D-glucose 2 g/l

Phenol-red 10 mg/l

-ACK (pH 7.4)

NH4Cl 8.3 g/l

KHCO3 1 g/l

Na2-EDTA 0.038 g/l

-Tyrode buffer (pH 7.4) for the degranulation assay

Tyrode 20x 25 ml

1m CaCl2 1ml

Hepes 2.38 g

Glucose 0.5 g

BSA 0.5 g

Distilled water Up to 500ml

-Tyrode buffer 20x for the degranulation assay

NaCl 160 g

KCl 4 g

NaH2PO4*H2O 1.1 g

MgCl2*6H2O 2.03 g

Distilled water Up to 1000ml

28

-Substrate solution (pH 4.5) for the degranulation assay

p-nitrophenyl-N-acetyl-β-D-glucosamine 1.3 mg/ml

Sodium-citrate 0.1 M

-STOP buffer (pH 10.75) for the degranulation assay

Glycin 7.519 g

Distilled water Up to 500ml

-PBS buffer (pH 7.4)

NaCl 8 g/l

KCl 0.2 g/l

Na2HPO4*H2O 1.4 g/l

KH2PO4 0.2 g/l

-TMB buffer (pH 5.5) for ELISA

Na-acetate 0.1 M

-Lysis buffer

HEPES 50 mM

NaF 100 mM

EDTA 10 mM

EGTA 2 mM

Na-pirophophate 10 mM

Glycerol 10 %

Triton-X-100 1 %

29

-Sample buffer (2x) for SDS-PAGE

Tris/HCl (pH 6.8) 125 mM

SDS (10%) 2 %

Glycerol 20%

Bromphenol Blue (4%) 0,001%

2-ME 2%

-Running buffer for SDS-PAGE (pH 8.3)

Tris 3 g/l

SDS (10%) 10 ml/l

Glycin 14.4 g/l

Distilled water Up to 1000ml

-Transfer buffer for Western blot

Tris 3 g/l

Glycin 14.4 g/l

Distilled water Up to 1000ml

-FACS buffer

PBS

FCS 1%

Na azide 0.1%

-TBS buffer

NaCl 137 mM

TRIS 20 mM

HCl 1 M

Distilled water Up to 1000ml

30

3.1.2. Antibodies

Name Host Origin

a-β-chain mouse R. Siraganian, Bethesda,

USA

a-extracellular domain of β-chain

goat Santa Cruz Biotechnology, Santa Cruz, CA

IgE mouse A. Licht, Rehovot, Israel

a-IgE rat Z. Eshhar, Rehovot, Israel

a-Lyn rabbit Transduction Laboratories, NH, USA

a-Fyn rabbit Santa Cruz Biotechnology, Santa Cruz, CA

a-phospho-ERK1/2 rabbit R. Segen, Rehovot, Israel

a-phospho-p38 rabbit R. Segen, Rehovot, Israel

a-phospho-c-raf rabbit Cell Signaling Tech., Beverly, MA

a-total p38 rabbit R. Segen, Rehovot, Israel

a-mouse-IgG-HRP rabbit DAKO, Glostrup, Denmark

a-rabbit-IgG-HRP goat DAKO, Glostrup, Denmark

a-κ/λ-Al647 goat J. Prechl, Budapest, Hungary

a-goat-IgG-Al647 chicken Invitrogene, Carlsbad, CA

a-biotin-FITC mouse Sigma Aldrich, St. Luis, MO

a-c-kit-biotin F(ab)2 BD PharMingen, San Diego, CA

a-PI3K rabbit Upstate, Billerica, MA

PT-66 (a-phospho-tyrosine)

mouse Sigma Aldrich, St. Luis, MO

3.1.3. Reagents

Name Origin

Tissue culture media, supplements Gibco, Grand Island, NY

TritonX-100 Sigma Aldrich, St. Luis, MO

p-nitrophenyl-N-acetyl-β-D glucosamine Sigma Aldrich, St. Luis, MO

31

Ionomycin Sigma Aldrich, St. Luis, MO

Propidium-iodide Sigma Aldrich, St. Luis, MO

2.4-dinitrobenzene sulphonic acid-conjugated bovine serum albumin (DNP11-BSA)

A. Licht, Rehovot, Israel

Cytochalasin D Sigma Aldrich, St. Luis, MO

Enhanced chemiluminescence reagent (ECL) Amersham, UK

Fluo-3/AM Calbiocham

Materials used for SDS-gel electrophoresis BioRad, CA, USA

3.1.4. Peptides synthesis and protein labelling

Peptides were synthesized by solid phase technique utilizing 'Boc chemistry’ on MBHA-

resin, purified and characterized by reversed phase HPLC and mass spectrometry. C3a9

(DCCNYITR) was labeled with Bodipy650/665 and A2IgE with Cy3, FITC as suggested by

the manufacturer (Amersham-Pharmacia, NJ, USA). Dimer forms of the peptides were

synthesized by disulphide cross-linking of the monomer forms. Peptides were used at the

indicated concentration for each experiment. As control the GARDGNEYI sequence or the

solution buffer, containing 0.2 % DMSO was employed.

Table IV. The sequences of the peptides

Peptide Code Sequence

C3a7 CCNYITELR

C3a9 DCCNYITR

DCS-Monomer DCSNYITR

DCS-Dimer DCSNYITR

DCSNYITR

DSC-Monomer DSCNYITR

DSC-Dimer DSCNYITR

DSCNYITR

Control GARDGNEYI

32

3.2. Methods

3.2.1. Cells

The RBL-2H3 cell line, originally obtained from Dr. Reuben Siraganian (NIH, Bethesda

MD) was maintained in Dulbecco’s Modified Essential Medium (DMEM) supplemented by

5% FCS in a humidified atmosphere with 5% CO2 at 37°C. For the experiments, cells were

harvested following 15 min incubation with 10 mM EDTA in DMEM.

3.2.2. Differentiation of mast cells from bone marrow derived precursors

BMMC were isolated from C57/Bl6 mice as described by Nagao et al. (99). After

removing the epiphysis of the femur, cells of the bone marrow were washed by GKN into a 50

ml centrifuge tube. Following centrifugation (1300 rpm/10 min JOUAN centrifuge) the cells

were incubated with 1 ml ACK for the lysis of erythrocytes. After washing with GKN cells

were filtered with a cell strainer (BD Pharmingen, 70 µm). Cells were maintained in medium

RPMI + 10% FCS + 20% IL-3 containing WEHI-3 cell supernatant. After 5 weeks, an

approximately 95% pure MC population was obtained, showing high expression levels of

FcεRI and stem cell factor receptor (c-kit), as measured by flow cytometry (Fig.10.).

Figure 10.: Differentiated MCs obtained from mouse bone marrow-derived cells

After 5 weeks the differentiated MCs express high level of FcεRI and c-kit. The homogeneity

of MC population was measured by flow cytometry.

33

3.2.3. Degranulation assessed by the β-hexosaminidase assay

Mediator secretion by MCs in response to stimulation by FcεRI clustering was monitored

by measuring the activity of the secreted granular enzyme β-hexosaminidase, as described

earlier (96). RBL-2H3 cells were sensitized with DNP-specific IgE. Cells were seeded into

96-well plate (1.5 x 105cells/well). After two hours, cells were washed with Tyrode buffer.

For each degranulation assay we activated the sensitized cells with different Ag

concentrations and based on the results we chose a suboptimal concentration for the

experiments with peptides. To study the effect of the C3a-derived peptides on Ag-induced

response, sensitized cells were pre-incubated with a concentration range of the various

peptides for 5 min at room temperature before exposure to suboptimal Ag concentrations

(Fig.11.). After 1 hour at 37oC 25µl supernatant of each sample was incubated with 45µl

Substrate solution for 45 min at 37oC. The reaction was stopped with 150µl Stop buffer and

samples were measured by photometry at 405nm. We consider a peptide for inhibitory

molecule if the amount of the degranulation in the presence of this molecule is below 60% of

the antigen response.

Figure 11.: The protocol for the degranulation assay

A: MCs sensitized with DNP-specific IgE were stimulated by DNP11-BSA for 1 hour at 37oC.

Activation of MCs was assessed by determining the activity of the released β-hexoseaminidase

enzyme from the supernatants. B: To examine the effect of complement-derived peptides on

34

degranulation, the sensitized cells were treated with peptides for 5 min at room temperature

before Ag challenge.

3.2.4. Immunoprecipitation and Western blotting

RBL-2H3 cells were grown in 10 cm dishes (8 x 106 cells/dish) and saturated with the

DNP-specific A2IgE at 37°C in DMEM overnight. After washing, they were treated with the

C3a-derived or control peptide for 5 min at RT, followed by FcεRI clustering by 5 ng/ml

DNP11-BSA (a suboptimal antigen-dose) at 37°C for the indicated time. The reaction was

terminated by washing 5 ml ice-cold PBS buffer and frozen in liquid nitrogen. Cells were

lysed with 500 µl lysis buffer + 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate,

protease inhibitor cocktail 1:200 per dish. The cells were scraped and lysates were centrifuged

at 15 000g for 15 min at 4oC. The protein content of the post-nuclear supernatants was

adjusted using Bradford-assay prior to precipitation by the indicated antibody coated beads

(10µl beads/sample). The proteins were eluted by sample buffer containing 2-

mercaptoethanol, separated by SDS-PAGE and electrotransfered onto a nitrocellulose

membrane. Blots were incubated with the indicated antibodies and visualized by ECL. For the

MAPK-pathway experiments we used whole cell lysate and the active forms of MAPKs were

detected by specific antibodies.

3.2.5. Laser Scanning Confocal Microscopy

RBL-2H3 cells were harvested and incubated with 5 µM of the Cy3-conjugated IgE for 20

min at 4oC. After washing the cells were treated with 200 µM Bodipy650/665-conjugated

C3a9 or control peptide for 5 min at RT, followed by activation with the antigen (50ng/ml

DNP11-BSA) for 30 min at 37oC. The activation was terminated with 500µl ice-cold FACS

buffer. Fluorescence signals were analyzed in the green (excitation by 543 nm He-Ne laser)

and red (excitation by 632 nm He-Ne laser) optical channels of an Olympus FLUOView500

laser scanning confocal microscope (Hamburg, Germany). The cells were optically sliced to

512 x 512 pixel sections with 0.5 µm thickness. Images were further processed by the IMAGE

J software.

35

3.2.6. Internalization assay

RBL-2H3 cells were harvested and incubated with 5 µM FITC-conjugated IgE for 20 min

at 4oC for monitoring the total amount of the IgE-receptor. After washing, cells were treated

with 200 µM C3a9 or control peptide for 5 min at RT before activation by antigen (50ng/ml

DNP11-BSA) for the indicated time at 37oC. The reaction was terminated by 500 µl ice-cold

FACS buffer. After washing, cells were incubated with Alexa-647-labeled anti-κ/λ for 20 min

at 4oC for the detection of the surface remaining FcεRI. The amount of total IgE (FITC) and

surface-remaining IgE (Alexa-647) was analyzed by flow cytometry. To asses the degree of

FcεRI internalization - which corresponds to the decrease in surface remaining IgE -, first the

mean fluorescence intensity (MFI) of the background (i.e. of cells incubated with Alexa-647-

labeled anti-κ/λ only) was substracted from the MFI of surface-IgE-bound cells, at each

indicated time-point. The obtained MFIabsolute value of stimulated cells at each time point was

then divided by the MFIabsolute of the control cells (t = 0), to obtain the percentage of IgE

remaining on the cell surface.

3.2.7. Flow cytometric detection of FcεRI-β-chain

RBL-2H3 cells were incubated with 200 µM C3a9 or control solution for 5 min at room

temperature before the detection of FcεRI-β-chain using an antibody specific to the

extracellular domain of the β-chain, for 20 min at 4oC. After washing, bound antibody was

detected with anti-goat-Al647 for 20 min at 4oC in dark. As control goat serum of the same

IgG concentration was used. Samples were analyzed by flow cytometry on a FACSCalibur

using CellQuestPro software (Becton Dickinson, NJ, USA).

3.2.8. Monitoring free cytosolic Ca2+ ion concentration

A suspension of 5 x 106 IgE-sensitized cells (RBL-2H3) in 0.5ml RPMI 1640 medium

was loaded with 5 µM fluo-3 AM indicator for 30 min at 37oC. After washing, samples of

cells (5 x 105 cells/ml) were incubated with 200 µM C3a9 or control for 5 min at RT. Antigen

(10ng/ml DNP11-BSA) was added to the cells 30 seconds after initation of flow cytometric

recording, and changes in free cytosolic calcium ion concentrations were followed in the time-

36

resolved mode of BD Bioscience FACSCalibur flow cytometer. Data acquistion and analysis

were performed with the CellQuest software (BD Biosciences).

3.2.9. Cytokine secretion assay

BMMCs or RBL-2H3 cells senzitized with DNP-specific IgE were seeded in 96-well plate

(2 x 105 cells/well) and incubated with antigen (3ng/ml or 5ng/ml DNP11-BSA) in the

presence or absence of C3a9 peptide or the control for 3 h and 24 h. The supernatants were

transferred into new 96-well plate and the level of secreted cytokines was measured by ELISA

kits (R&D) according to the manufacturer’s instructions.

3.2.10. Passive Systemic Anaphylaxis (PSA) and measurment of histamine in the serum of treated mice

Mice (C57/Bl6) were sensitizied by injecting 3 mgs of DNP-specific mouse IgE mAb in

the retro-orbital vein. 24 h later the animals were treated by intranasal application of the

peptides in drops. After 10 min mice were challenged with i.v. injection of 0,5 mg HSA-DNP

per mouse. Blood histamine levels were determined by ELISA (Beckman Coulter IM2015) 5

min after antigen challenge according to the manufacturer’s instructions.

3.2.11. Basophil activation test

Activation of human basophil granulocytes was measured by using the Flow-Cast

Basophil Activation Test (BAT) (Bühlmann) according to the manufacturer’s instructions.

The protocol (Fig.12.) is as follows: Blood sample treated with K-EDTA as anticoagulant was

centrifuged for 5 min at 200g to separate the leukocyte-containing plasma and the fraction of

erythrocytes. The plasma-fraction was centrifuged for 10 min at 500g, and 110µl Stimulation

buffer (containing heparine and IL-3) was added to the pellet. For measuring the effect of the

peptides, 25µl leukocyte-suspension was incubated with the indicated concentrations of

peptides for 5 min at room temperature. Cells were activated with a suboptimal amount of

anti-FcεRI (40x dilution of the anti-FcεRI preparation provided with the kit; the concentration

of the antibody is unknown) for 40 min at 37oC. Then 25µl blocking buffer and 10µl Staining

reagent (anti-IgE-FITC and anti-CD63-PE) were added to the cells and incubated for 30min at

37

4oC. After this 1.5ml Lysis buffer was added to the cells for 5 min at room temperature and

the reaction was stopped with 500µl Blocking buffer. After centrifugation, the supernatant

was decanted and the cell pellet was resuspended with 200 µl Blocking buffer. Samples were

analyzed by flow cytometry on a FACSCalibur using CellQuestPro software (Becton

Dickinson, NJ, USA) (Fig.13.).

Figure 12.: The steps of the FLOW-CAST Basophil Activation Test (BAT) Flow Cytometry

/Bühlmann/ kit

38

A B C

Figure 13.: Detection of CD63 expression on human basophil granulocytes

A: Gating on the lymphocytes and basophil granulocytes B: Gating on the IgE-positive cells

present in the population of lymphocytes + basophil granulocytes. C: Detection of CD63-

positive human basophil granulocytes in the upper-right quadrate of the dot-plot

3.2.12. Statistical analyses

Differences between data were analyzed with impaired Student’s t-test. The significant

differences between histograms was calculated with the Kolomogrov-Smirnov (K-S) Two-

Sample Test.

39

4.Results

4.1. The complement-derived inhibitory peptide C3a9 colocalizes with antigen-clustered FcεRI on MCs

We have earlier shown that the complement-derived component C3a and its derived

peptides C3a7 and C3a9 inhibit FcεRI-induced degranulation of MCs and bind to the FcεRI

β-chain on unperturbed cells (96,98). As the β-subunit has earlier been shown to act as an

amplifier of the IgE-mediated signaling (50), we investigated whether the inhibitory peptide

remains bound to the receptor-complex even after FcεRI-IgE clustering with the antigen,

thereby maintaining the inhibitory activity. RBL-2H3 cells were incubated with Cy3-

conjugated anti-DNP IgE for 20 min at 4oC and after washing, they were treated with

Bodipy650/665-conjugated C3a9 peptide for 5 min at RT. Following this, IgE-bound FcεRIs

were clustered by DNP11-BSA for 30 min at 37oC. As shown in Figure 14. part of the peptide

remains colocalized with the FcεRI and its majority is co-internalized with the aggregated

FcεRI-IgE during this time period. Bodipy650/665-conjugated control peptide does not bind

to the RBL-2H3 cells (data not shown).

Figure 14.: C3a9 binds to both unperturbed and stimulated MCs

Confocal microscopic images of RBL-2H3 cells labeled with Cy3-IgE and Bodipy650/665-

C3a9. Cells were either left untreated as control (Cont.) or stimulated by antigen for 30 min

(Ag). Equatorial slices are shown for a representative cell. Cy3 fluorescence images (left),

40

Bodipy650/665 fluorescence images (middle), and their overlay (right) are displayed. One

representive experiment of three is shown.

4.2. C3a9 binds to the extracellular domain of FcεRI-β-chain

The β-chain of the high affinity IgE-receptor is known to amplify the FcεRI-coupling

network (50,51) and interestingly this ITAM-containing receptor chain also has an inhibitory

function (100). As shown in Fig.14. we found that the inhibitory peptide colocalizes with

antigen-clustered FcεRI on MCs on both the unperturbed and stimulated cells, moreover,

earlier we demonstrated by Surface Plasmon Resonance measurement that C3a9 binds to the

β-chain (96,98). It was important to see if the interaction of the peptide with the β-chain

occurs on living cells, as well. To test this, after treatment of RBL-2H3 cells with peptide

C3a9 they were incubated with an antibody recognizing the extracellular domain of the β-

chain. Since binding of the anti-β chain antibody was detected by Alexa647 labeled anti-goat-

antibody, we used goat serum as a control. As shown in Figure 15, the availability of the β

chain is inhibited in the presence of the peptide. Table V. shows the values of Gmean of

histograms.

Figure 15.: C3a9 binds to the FcεRI-β-chain

Cells were incubated with or without peptide C3a9. Binding of the goat antibody specific to

the extracellular domain of the β-chain was detected by anti-goat-Al647. We used normal

goat serum as a control. One representative experiment of three is shown.

41

Table V. Inhibition of extracellular domain of β-chain binding to the MCs by C3a9

* p=0,001

Name of histogram Geometric Mean

Control 9,69

Absence of C3a9 20,51

Presence of C3a9 15,25 *

4.3. Treatment of MCs with C3a9 enhances the internalization of FcεRI after receptor clustering

FcεRI has been found to rapidly migrate to the lipid raft and endocytosed after its

clustering (58,101,102). As shown above the inhibitory peptide remains bound to the FcεRI

upon internalization (Fig.14.), therefore we investigated the effect of C3a9 on the endocytosis

of the receptor. We monitored the total amount of FcεRI by FITC-labeled IgE and the amount

of IgE remaining on the surface after FcεRI-clustering by Alexa647-labeled anti-κ/λ. As

shown in Figure 16., binding of C3a9 significantly enhances the receptor endocytosis already

at 5 min after clustering, and this effect subsides to the control level at 30 min. This result

demonstrates that C3a9 enhances the internalization of FcεRI in the time-interval where

intracellular signaling steps are initiated. The treatment with C3a9 may lead to receptor-

recycling to the cell surface, instead of being degraded at the beginning of endocytosis,

however this process still needs to be investigated.

42

Figure 16.: The effect of C3a9 on FcεRI internalization

The high affinity IgE receptors were cross-linked on IgE-sensitized RBL-2H3 cells with

DNP11-BSA for the indicated time-points at 37oC in the presence or the absence of C3a9. In

the control sample dilution buffer containing 0.2% DMSO was added to the cells. FcεRIs

remaining on the cell surface were detected by Alexa647-labeled anti-κ/λ antibody. For the

expression of FcεRI cells were analyzed by flow cytometer. (A) Measurement of the total

amount of FcεRI (both cell surface and intracellular) as a function of time. (B) The expression

of the FcεRI on the cell surface after clustering, as a function of time. Calculation of the

percentage of FcεRI is described in the section of Material and Methods. Statistical analysis

of the histograms shows a significant difference * p=0.01. One representative experiment of

three is shown.

43

4.4. Binding of C3a9 to MCs causes dissociation of the FcεRI-β-subunit bound PTK Lyn and Fyn

It is well-documented that the association of Lyn with the FcεRI β-chain is a key step in

the initiation of the stimulus-response coupling network in MCs (48). It has also been reported

that Fyn, another src family PTK, has a complementary role in MC activation (67). In order to

further resolve the mechanism of the inhibitory process in molecular detail, we investigated

how C3a9 affects the above events. Since we found that C3a9 binds to the FcεRI on both

unperturbed and stimulated cells (Fig.14.), we studied whether the peptide affects the

association of the FcεRI-β-chain with these two PTKs, Lyn and Fyn. As shown in Figure 17,

the presence of C3a9 causes an almost complete reduction of the FcεRI-β-chain associated

Lyn and exerts a somewhat slighter effect on the association between Fyn and the receptor.

These results demonstrate that the peptide exerts its inhibition already at the earliest steps of

the FcεRI-clustering induced coupling network in MCs.

Figure 17.: C3a9 inhibits the association of the β-chain with Lyn and Fyn in MCs triggered

by FcεRI-clustering.

RBL-2H3 cells (1 x 107/sample) were either left untreated (C), FcεRI-stimulated for 30 sec

(Ag), treated with C3a9 for 5 min and then activated through the FcεRI (C3a9 + Ag) or

incubated in DMSO-containing dilution buffer (Contr.+Ag). After treatment cells were lysed

and samples containing equal protein amounts were immunprecipitated employing an IgE-

specific antibody. Samples were analyzed by sequential Western blotting (WB) with antibodies

specific to Lyn, Fyn and β-chain. Detection was carried out by ECL. One representative

44

experiment of three is shown. Numbers indicate fold induction of the two Src PTK associated

to the FcεRI normalized to the amount of FcεRI-β-chain.

4.5. C3a9 inhibits the phosphorylation of several intracellular proteins including Lyn, the β subunit of FcεRI and PI3K

Based on our results shown above (Fig.17.) next we investigated the effect of C3a9 and

also of C3a7, a natural sequence of the complement protein C3a on the tyrosine

phosphorylation of intracellular proteins. IgE-sensitized RBL-2H3 cells were preincubated

with the peptides at 200µM for 5 min at room temperature before activation with Ag (5ng/ml

DNP11-BSA) for 2 min at 37oC. Fig.18.A. shows that the FcεRI clustering-induced

enhancement of protein tyrosin phosphorylation of several intracellular proteins is markedly

reduced in the presence of the C3a-derived peptides (C3a7 and C3a9). In the intracellular

signaling process after the association of Lyn src kinase and the β-chain, the active Lyn

transphosphorylates more Lyn kinases, the ITAMs of the β-chain and the γ- dimers (62,103),

which serve as a docking site for other SH2-domain containing signaling molecules (49,104).

As Fig.18.B shows C3a7 and C3a9 inhibit the phosphorylation of Lyn and also the β-chain.

Since PI3K has a central role in the regulation of FcεRI-coupling network and MC secretory

response (105), we investigated the effect of complement-derived peptides on the activation of

PI3K, as well. As shown in Fig.18.B both C3a7 and C3a9 strongly inhibit the phosphorylation

of this enzyme (98). It has to be mentioned that these peptides did not cause by themselves

any change in the pattern of tyrosine phosphorylated proteins of RBL-2H3 cells (data not

shown).

45

Figure 18.: C3a7 and C3a9 inhibit tyrosine phosphorylation of Lyn, the β subunit of FcεRI

and PI3K

IgE-sensitized adherent RBL-2H3 cells were incubated for 5 min with C3a7, C3a9 or the

control peptide before antigenic challenge at 200µM concentration. Cells were then lysed and

immunoprecipitated with antiphosphotyrosine Ab PT-66. Samples are as follows: Lane 1:

non-stimulated cells; lane 2: antigen-stimulated cells; lane 3: C3a7-pretreated cells, lane 4:

C3a9-pretreated cells, lane 5: cells pretreated with control peptide. A: General

phosphorylation pattern of RBL-2H3 cells. Western blot was developed by PT-66 antibody. B:

Phosphorylation of Lyn, β-chain of FcεRI and PI3K. Western blots were developed with

antibodies reacting with Lyn, the β-chain and PI3K specific antibody, respectively. The

membrane was immunoblotted with anti-actin to confirm that equal amounts were loaded.

One representative experiment of five is shown in each panel.

4.6. The effect of C3a9 on the elevation of [Ca2+]i

The aggregation of FcεRIs causes an increase of [Ca2+]i level, which is crucial for the

activation of the Ca2+-dependent protein kinase C (PKC) and for the degranulation of MCs.

Several adaptors and enzymes are known to have an important role in the regulation of the

Ca2+-response such as the Bruton’s tyrosine kinase (Btk) or the phosphatidylinositol 3-kinase

(PI3K). The activation of these molecules depends on the src kinases (Lyn and Fyn) and Syk

(104). Figure 17. demonstrates that treatment with peptide C3a9 inhibits the earliest step of

46

the receptor-coupling network. In Figure 19 we show that the antigen-induced elevation of

[Ca2+]i is also decreased in the presence of C3a9.

Figure 19.: The effect of C3a9 on the elevation of [Ca2+]i

RBL-2H3 cells were sensitized with DNP-specific IgE and loaded with fluo-3/AM (5µM) for

30 min at 37oC. After washing, the cells were preincubated with 200µM peptide C3a9 or

control peptide for 5 min at room temperature. At 30 seconds, antigen (10 ng/ml DNP11-BSA)

was added to the cells and fluorescence intensity was monitored as a function of time. One

representative experiment of three is shown.

4.7. C3a9 inhibits the MAPK-pathway

Activated MCs are known to release several cytokines, including IL-4, IL-5, IL-9, IL-13

and TNF-α, which are important in generating an inflammatory response (106). It has been

shown that FcεRI clustering leads to the activation of MAPKs, namely ERK1/2, p38 and JNK

in MCs which are known to play a role in both the induction and regulation of cytokine

expression (107-109). Now we investigated whether binding of C3a9 to FcεRI has any effect

47

on the MAPK-pathway. As illustrated in Fig. 20. the complement-derived peptide strongly

inhibits the phosphorylation of ERK1/2 and slightly affects the activation of p38. As the main

activation route of ERK is via the ras-raf-MEK pathway (107,110), we next examined

whether C3a9 interferes with the phosphorylation of raf. As shown in Fig.20. the peptide does

not affect the raf phosphorylation, suggesting that C3a9 inhibits ERK activation via an

alternative route.

Figure 20.: C3a9 inhibits the MAPK-pathway in MCs triggered by FcεRI-clustering

RBL-2H3 cells (1 x 107/sample) were either left untreated (C), FcεRI-stimulated for 5 min

(Ag), treated with C3a9 for 5 min and then activated through the FcεRI (C3a9 + Ag) or

pretreated with control peptide (Contr.+Ag). Cells were lysed and samples containing equal

protein amounts were analyzed by WB using antibodies specific to the active forms of ERK,

p38 and c-raf. Detection was carried out by ECL. One representative experiment of three is

shown. Numbers indicate fold induction of the phosphorylation of ERK, p38, c-raf normalized

to the total amount of p38.

48

4.8. C3a9 inhibits secretion of the inflammatory cytokines

As we found that C3a9 inhibits the activation of two members of the MAPK-family

(Fig.20.), we aimed to clarify whether the production of inflammatory cytokines is also

affected. The expression of the inflammatory cytokines, TNF-α and IL-6, is regulated by the

MAPK-pathway (111,112), therefore we investigated the effect of C3a9 on the release of

these cytokines. First we measured the release of TNF-α by RBL-2H3 after 24h in the

presence of C3a9 and C3a7. As Fig.21. shows the two C3a-derived peptides (C3a7 and C3a9)

inhibit the secretion of TNF-α while the peptides have no effect on the secretion alone (98).

As it is important to know how the cytokine production of MCs of physiological origin is

effected by C3a9 we set out to investigate the peptide’s affect on the FcεRI-clustering induced

secretion of TNF-α and IL-6 by bone marrow derived mast cells (BMMCs). To study the

time-course of the process, supernatants were taken after stimulating the cells by DNP11-BSA

antigen in the presence or absence of C3a9 for 3 and 24 hours. As illustrated by Figure 22,

C3a9 inhibits the secretion of both IL-6 and TNF-α. The results show that after 24 hours the

inhibition is more pronounced, suggesting that the inhibitory mechanism also involves the

gene transcription process.

Figure 21.: The inhibitory capacity of synthetic peptides C3a7 and C3a9 on the cytokine

production of RBL-2H3 cells.

RBL-2H3 cells were saturated with DNP-specific IgE and preincubated with the indicated

concentrations of C3a-derived peptides for 5 min at room temperature, followed by a

49

suboptimal antigen challenge (5ng/ml DNP11-BSA). Secretion of TNF-α cytokine was

measured by ELISA. One representative experiment of three is shown. The error bars

represent SD of triplicate samples.

Figure 22.: C3a9 inhibits the antigen-induced secretion of TNF-α and IL-6 by BMMC

IgE-sensitized BMMCs (2 x 105/ well) were incubated with a suboptimal amount of the

antigen (3 ng/ml DNP11-BSA) for 3 and 24 h in the presence of C3a9 or control (200µM).

Secretion of TNF-α and IL-6 was assessed by ELISA. White column: cytokine secretion of

C3a9-treated cells; black column: control samples. One representative experiment of three is

shown. The error bars represent SD of triplicate samples. * p<0,01, **p<0,005

4.9. F-actin does not play a role in the C3a9 induced inhibition

It is well described that F-actin regulates the early and the late events in the FcεRI -

coupling network (84). Cytochalasin D, which inhibits actin polymerization by capping the

barbed end of actin filaments and prevents their elongation (113), was reported to enhance the

IgE-mediated tyrosine phophorylation and Ca2+-response of MCs and it was also shown to

allow a more extensive and sustained association between FcεRI and raft markers (85). F-

actin negatively regulates the phosphorylation of Syk and Lyn interaction with FcεRI (86).

50

We have earlier proven that C3a and its deritatives inhibit the immediate activation of MCs

(96-98). In the previous paragraphs we have shown that the more effective peptide, C3a9 is

able to interrupt the earliest steps of the FcεRI-induced signaling and inhibits the late phase

activation of MCs, as well. Based on these findings we thought it worth to investigate whether

Cytochalasin D affects the inhibitory mechanism.

To this end IgE-sensitized RBL-2H3 cells were pretreated with 2µM Cytochalasin D for

10 min at 37oC. After washing, cells were preincubated with the peptides before the

suboptimal antigen challenge. The activity of released β-hexoseaminidase in the samples in

Figure 23. illustrates that the inhibition of actin polymerization does not alter the effect of

C3a9 on the IgE-mediated degranulation (Fig.23.). These data suggest that F-actin has no role

in the inhibitory effect of the complement-derived peptide C3a9.

Figure 23.: F-actin has no role in the inhibitory mechanism of C3a9.

IgE-sensitized cells were treated with 2µM Cytochalasin D for 10min at 37oC. Cells were

preincubated with the peptides at the indicated concentrations for 5min at room temperature

before activation with the suboptimal dose of the antigen (DNP11-BSA). Release of the

granular enzyme, β-hexoseaminidase was measured. One representative experiment of three

is shown. The error bars represent SD of triplicate samples.

51

4.10. Inhibition of Passive Systemic Anaphylaxis (PSA) in mice pretreated with the complement-derived peptide

Our in vitro studies clearly show that the C3a-derived peptide C3a9 inhibits the IgE-

mediated activation of both the rat mucosal mast cell-line (RBL-2H3) and mouse bone

marrow-derived mast cells (BMMC) very efficiently (98). The next aim was to see the effect

of the peptide in an in vivo mouse model of passive systemic anaphylaxis (PSA).

Mice were passively sensitized with anti-DNP IgE and 24 hours later the animals were

treated with the peptides as described in the section of Materials and methods. 10 minutes

later mice were challenged with DNP-HSA and after 5 minutes blood was taken from the

animals and plasma histamine levels were measured by ELISA. As illustrated in Figure 24.

there is a significant decrease in the plasma histamine level of the group of animals pretreated

with the peptide C3a9 (Fig.24.).

As the increase of histamine in plasma has been shown to correlate with systemic

anaphylaxis (114), based on our results we suggest that C3a9 is a potent inhibitor of the

generalized hypersensitivity reaction in mice.

Figure 24.: C3a9 inhibits the IgE-mediated histamine release in vivo.

Mice sensitized with antigen-specific IgE were pretreated 24 hours later for 5 minutes with

C3a9 (white) or control solution (black). Then animals were challenged by i.v. injection of

HSA-DNP. Histamine levels were determined by ELISA in plasma collected from mice 5 min

after challenge. One representative experiment of three is shown. * p<0,05

52

4.11. Testing newly designed peptides and peptidomimetics on the degranulation of RBL-2H3

As one of our goals is to develop anti-allergic substances for therapeutic use, we tested the

effect of several peptides and peptidomimetics on MC degranulation, aiming to find more

effective and stable inhibitory molecules, based on the sequence of C3a9 (DCCNYITR).

These peptides and peptidomimetics were designed and synthesized by Dr. Gábor Tóth

(University of Szeged). IgE-sensitized cells were incubated with the synthetic peptides for 5

min at room temperature before the stimulation with a suboptimal Ag concentration. Then the

activity of the secreted granular enzyme, β-hexoseaminidase, was measured..Until now we

could not identify a substantially more effective molecule than the C3a9 peptide. Importantly

however, we have proven that the two cysteins present in this peptide have a crucial role in

exerting the inhibition of MC triggering. We found that dimers generated based on the

sequence of C3a9 by replacing one of the cysteins to serine, ie. the DCS-dimer: (disulphide-

linked DCSNYITR) and the DSC-dimer: (disulphide-linked DSCNYITR) are more effective

than the monomeric forms of the same sequences; i.e. the DCS-monomer: (DCSNYITR) and

the DSC-monomer (DSCNYITR) (Fig.25.). We also investigated the effect of the sequence,

where the two Cys had been replaced by Ser (DSSNYITR), but this peptide had no inhibitory

effect at all on the degranulation of MCs (data not shown). Based on these data we assume

that C3a9 acquires its strong inhibitory activity in solution by forming dimers via disulphide-

links.

53

Figure 25.: The effect of monomeric and dimeric peptides on the degranulation by RBL-2H3.

The following peptides were used: C3a9: DCCNYITR; DCS-dimer: disulphide-linked

DCSNYITR; DCS-monomer: DCSNYITR; DSC-dimer: disulphide-linked DSCNYITR; DSC-

monomer: DSCNYITR. Cells were sensitized with DNP-specific IgE and preincubated with

the indicated concentrations of peptides for 5 min at room temperature, followed by a

suboptimal Ag (DNP11-BSA) challenge. Release of the granular enzyme, β-hexoseaminidase

was measured. Data are presented as net percentages of the enzyme release of the cells

treated with control before Ag challenge. Columns represent the average of 3 independent

experiments and error bars represent SD.

4.12. The effect of monomeric and dimeric forms of C3a9-derived peptides in Passive Systemic Anaphylaxis (PSA)

Next we investigated how the synthetic dimers generated based on the sequence of C3a9

influence the in vivo anapylaxis. We used the mouse model of passive systemic anaphylaxis

(PSA, see details in the section of Materials and Methods and also in 4.10.) to assess the

peptides’ effect on MC activation in vivo. As shown in Figure 26. all the peptides which

inhibit the degranulation of RBL-2H3 in vitro (Fig.25.), also blocks the IgE-mediated

histamine release in vivo. It has to be pointed out that in this in vivo assay the monomeric

forms (DCS-Monomer and DSC-Monomer) inhibited the allergic reaction to a similar extent

as the dimers. Although at present we can not explain this result, we suppose that in this case

54

dimerization of the peptides might have occured after their administration in the mice – most

probably on the mucosal surface.

Figure 26.: IgE-mediated histamine release in the presence of syntetic peptides.

The following peptides were used: Control: GARDGNEYI; C3a9: DCCNYITR; DCS-dimer:

disulphide-linked DCSNYITR; DCS-monomer: DCSNYITR; DSC-dimer: disulphide-linked

DSCNYITR; DSC-monomer: DSCNYITR. Mice were sensitized by i.v. injection of IgE in the

retro-orbital vein. 24 h later the animals were challenged with i.v. injection of HSA-DNP in

the presence of the indicated peptides (200µM). Histamine levels were determined by ELISA

in plasma collected 5 min after challenge. Columns represent the average of 3 independent

experiments and error bars represent SD.

4.13. The effect of monomeric and dimeric forms of C3a9-derived peptides on the activation of human basophil granulocytes

As demonstrated above (Fig.25., 26.), we have shown that the complement-derived

peptides inhibit the activation of rodent MCs both in vitro and in vivo. Since we aim to

develop anti-allergic substances for human therapeutic use, next we set out to investigate the

effect of these peptides on human basophil granulocytes, the circulating equivalent of MCs.

Basophils also express FcεRI, contain plasma granules, and upon triggering they release

histamin and several other mediators of the allergic reaction (16,115). The activation of

human basophil granulocytes was followed by flow cytometric detection of the expression of

CD63 (116,117). CD63 belongs to the tetraspanin family and in the resting basophils this

55

molecule is located on the membrane of the granules. After activation of the cells via FcεRI

clustering the expression of CD63 increases on the cell surface (118).

As detailed in the section of Materials and Methods, basophil granulocytes were

preincubated with the complement-derived peptides for 5 min at room temperature before

their activation with a suboptimal concentration of anti-FcεRI antibody for 40 min at 37oC.

We identified basophils by staining with anti-IgE-FITC and followed their activation using

anti-CD63-PE antibody. As the results of flow cytometric measurements show in Fig. 27,

C3a9 and also the dimeric forms of its derivatives (i.e. the DCS-Dimer and the DSC-Dimer),

inhibit the activation of the basophil granulocytes. The monomeric forms of the synthetic

peptides however, exert a weaker inhibition (Fig.27.). This result clearly shows that C3a9

(DCCNYITR) and its dimeric derivatives are potent inhibitors of the IgE-mediated activation

of human basophils, as well. Thus, these molecules are good candidates for further

development of anti-allergic drugs.

Figure 27.: The effect of complement-derived peptides on the activation of human basophil

granulocytes.

The following peptides were used: C3a9: DCCNYITR; DCS-dimer: disulphide-linked

DCSNYITR; DCS-monomer: DCSNYITR; DSC-dimer: disulphide-linked DSCNYITR; DSC-

monomer: DSCNYITR. Cells were preincubated with the indicated concentrations of

peptides before the activation by the FcεRI-specific antibody. The activation of basophil

granulocytes – which correlates with the increased expression of CD63 - was followed by

flow cytometry. Data are presented as net percentages of the CD63 expression of the cells

56

treated with control before activation. Columns represent the average of 3 independent

experiments and error bars represent SD.

4.14. The effect of commercially available anti-allergic drugs on the activation of human basophil granulocytes; comparison with peptide C3a9

In order to get information on the inhibitory capacity of the C3a9 peptide, we compared its

effect with the commercially available anti-allergic substances, such as cromolyn sodium,

hydrocortison and dexamethason.

We assesed the IgE-mediated activation of human basophils in the presence of C3a9 and

the anti-allergic drugs by flow cytometry (see details above in Matherials and Methods and

4.13.). As illustrated in Fig. 28. none of the commercial anti-allergic drugs were better

inhibitors of basophil activation than C3a9 in our experimental system.

Figure 28.: The effect of C3a9 and commercial anti-allergic drugs on the activation of

human basophils.

Cells were preincubated with the indicated concentrations of peptide C3a9 and the

commercially available anti-allergic drugs before the activation by the FcεRI-specific

antibody and the activation of human basophils are followed by flow cytometry. Data are

57

presented as net percentages of the CD63 expression of the cells treated with DMSO-

containing solution buffer as a control before activation. One representative experiment of

three is shown.

58

Our major results

Investigating the molecular mechanism of the inhibition of FcεRI-clustering induced

activation of MCs by complement-derived peptides we have proven the following:

● C3a9 binds to FcεRI-β-chain on both unperturbed and activated MCs.

● C3a9 enhances the antigen induced internalization of FcεRI.

● C3a9 severs the interaction between Lyn, Fyn src kinases and the β subunit of FcεRI.

● C3a7 and C3a9 inhibit the phosphorylation of Lyn, the β subunit of FcεRI and PI3K.

● C3a7 and C3a9 decrease the IgE-mediated phosphorylation of several intracellular

proteins.

● C3a9 inhibits the elevation of [Ca2+]i.

● C3a9 inhibits the activation of MAPK-pathway.

● The peptides decrease the production of inflammatory cytokines by mast cells.

● F-actin has no role in the inhibitory mechanism caused by C3a9.

● C3a9 inhibits systemic anaphylaxis in a mouse model.

● C3a9 exerts its inhibition most probably by its dimeric forms.

Figure 29.: Points where the inhibitory effect of C3a9 have been demonstrated.

C3a9 binds to the β-chain of FcεRI and inhibits the earliest step of the signaling pathway (i.e.

the association of Lyn and Fyn src kinases with the β-chain of FcεRI). As a consequence, both

the immediate (degranulation) and late phase activation (cytokine production) of MCs are

blocked. In this scheme of FcεRI-coupling network the red symbols ( ┬) indicate these points.

59

5. Discussion

The secretory response of MCs is triggered by FcεRI-dependent or independent pathways.

It is known that the complement activation products C3a and C5a, the so-called

anaphylatoxins, induce the secretory response of serosal type MCs, while the mucosal type

ones do not respond to these stimulants (12,119). We had previously shown that C3a inhibits

the FcεRI-mediated activation of MCs of the RBL-2H3-line, while C5a does not, and it had

also been demonstrated that C3a binds to the β-chain of FcεRI (96). The inhibitory sequence

of this complement factor had been identified (amino acids 56-64; CCNYITELR), and the

nonapeptide was designated C3a7. We had proven that this sequence does not contain the

stretch of C3a which is responsible for the activation of serosal type MCs via binding to C3a

receptors (97). Later we have reported that C3a7, as well as its modified derivative C3a9

(DCCNYITR), inhibit the degranulation of mucosal type MCs (RBL-2H3 cell line and

BMMCs) and also of the serosal type ones (isolated rat peritoneal mast cells) by binding to

the FcεRI-β-chain (98).

The role of the β-chain

The β subunit of FcεRI is expressed only on the surface of MCs and basophil

granulocytes, while its trimeric form, lacking the β-chain, appears on the surface of several

other human cell types, such as Langerhans cells, monocytes and macrophages. This subunit

of the receptor was found to play a crucial role for neither receptor expression nor FcεRI-

coupling network (47), but has earlier been reported to act as an amplifier of the FcεRI

stimulus; moreover an association between atopy and structure variants of this chain has also

been reported (50,120). On the other hand Furomoto et al. have described that the FcεRI-β-

chain may function to dampen degranulation or cytokine secretion (100), i.e. it has also a

negative modulatory role in MC activation. This modulatory function of the β-chain might be

explained by its structure. Namely, the ITAM of this receptor-chain possesses a noncanonical

tyrosine residue (Y225) that is situated between the two canonical tyrosines (Y219 and Y229)

found in conventional ITAMs (Fig.30.) (49). Y219 is responsible for the association between

the β-chain and Lyn src kinase while the other canonical Y229 has no role in this process. The

lack of Y219 phosphorylation leads to the inhibition of degranulation and cytokine

production. By contrast, the mutation of the noncanonical tyrosine (Y225) caused a marked

60

increase in MC cytokine production (IL-6 and IL-13) without affecting degranulation and

lipid mediator secretion. This tyrosine also plays an important role in the regulation of NFκB

activation (100). Thus it appears that the ITAM of the β-chain can also promote negative

signals (49).

Now we have shown that the complement-derived peptide C3a9 binds to the FcεRI-β-

chain on unperturbed cells and maintains binding to the FcεRI-IgE also after aggregation by

antigen and following internalization. Our results led us to assume that C3a and its derivatives

regulate the IgE-mediated stimulation of MCs upon binding to the β-chain extracellular loops,

clearly illustrating its function as a modulator.

Figure 30.: Structure of FcεRI illustrating its newly defined inhibitory motifs.

The FcεRI-β-chain posseses ITAM in its intracellular region. This activation motif contains a

noncanonical tyrosine residue at position 225, which is involved in the negative regulation of

cytokine production by MCs (49).

61

Receptor endocytosis as a potential regulator of MC activation

It has been shown recently that receptor endocytosis is an important regulatory step of the

signaling process (79), therefore we investigated the effect of C3a9 on the internalization of

the FcεRI. We found that in the presence of the peptide the rate of the antigen-clustered

receptors’ endocytosis is significantly enhanced. Our results clearly demonstrate that this

increase occurs in the first few minutes after receptor-clustering, a time period known to be

crucial for the initiation of effector functions of MCs. As Fig.16. illustrates the expression of

FcεRI on the cell surface after 30 min of antigen challenge is similar in the presence or

absence of C3a9. This suggests that the peptide might protect the internalized receptor from

degradation; however this possibility needs further investigation.

The mechanism of FcεRI internalization is still debated. While Wilson at al. have reported

that the endocytosis of antigen cross-linked receptors is clathrin-mediated, Fattakhova et al.

have shown that this process depends on the raft-association of FcεRI (101,102). Inasmuch

as the endocytosis of aggregated FcεRIs takes place in a few minutes, this process could be

mediated by lipid rafts as an early event, while the clathrin-mediated endocytosis might also

take place later upon receptor activation (101). Moreover, it is still an open question what

mechanisms drive FcεRIs to recycling or degradation. The antigen-clustered FcεRIs

translocate to the lipid rafts where they are ubiquitinated by Cbl ubiquitin ligase. This process

can direct the internalized receptors to the endosomal or lysosomal pathway (77).

Ubiquitination could act as a signal triggering lateral removal of the receptors from lipid rafts,

thereby limiting the duration of the signaling response (77). Cbl has been also described as an

inhibitor of the activation of PTKs such as Syk, and the cytokine production by MCs (78,80).

Nedd4, another ubiquitin ligase, can also ubiquitinate the aggregated FcεRI and direct it to the

clathrin-coated membrane region where the clustered receptors can be endocytosed (121).

CIN85, which is a scaffold protein, has been described to have an important role in the

internalization of FcεRI and to negatively regulate the receptor’s response coupling network

(79). Association of cross-linked FcεRI with clathrin-coated pits is regulated not only by

ubiquitination and binding the CIN85 adaptor molecule (122), but the β subunit may have also

a role in this process (123). The ITAMs of FcεRI have a role in receptor internalization as

well, however the precise function of the tyrosine residues is still unclear (124). It has been

described that inhibition of phosphatases which results in an increased tyrosine

phosphorylation of the receptors, leads to decreased receptor internalization (125). As

treatment of MCs with C3a9 enhances the receptors’ endocytosis in the first 5 minutes, this

62

process most probably depends on lipid rafts where the receptors can be ubiquitinated. Thus

the C3a-derived peptide may cause the inhibition of various MC functions by this way.

The FcεRI-coupling network

In view of these results, we further examined the effect of complement-derived peptide on

the association between the FcεRI-β-chain and the two src PTKs, Lyn and Fyn. We found that

binding of C3a9 clearly impairs these interactions. The src family Lyn kinase has long been

known to serve as a major element in the initiation and regulation of the signal transduction

through FcεRI. Upon FcεRI clustering, the receptor-associated Lyn transphosphorylates itself,

thereby undergoing activation as well. It also transphosphorylates the ITAMs on both β- and

γ-chains. While the β-chain functions as a binding site for Lyn, the phosphorylated γ-chain

becomes a docking site for the PTK Syk through its SH2 domain which then also undergoes

activation. These important activation and signal amplification processes are followed by

recruitment of additional enzymes and several adaptor molecules leading to the formation of

signaling complexes (48,49,126).

Parravicini et al. have described the role of Fyn, another src family member PTK in the

FcεRI coupling network of MCs (67). Fyn serves an alternative to Lyn in the FcεRI -mediated

signaling and is responsible for the phosphorylation of Gab2 directly and for the activation of

PI3K and Akt leading to degranulation indirectly (66,67). Fyn also associates with the FcεRI-

β-chain in a similar manner to Lyn and phosphorylates the adaptor molecule Gab2, which

binds PI3K (127). Gomez at al. have described that Fyn src kinase also regulates the

activation of p38 and JNK, while it has no effect on the phosphorylation of ERK. This src

kinase affects the cytokine production by MCs, as well (68). We have earlier shown that the

C3a-derived peptides inhibit the FcεRI-mediated immediate secretory response of both serosal

and mucosal-type MCs (98). Here we demonstrate that binding of C3a9 to MCs strongly

reduces the interaction of Lyn and Fyn with the FcεRI-β-chain. We show that the

complement-derived peptides C3a7 and C3a9 inhibit the tyrosine phosphorylation of FcεRI-β-

chain and Lyn kinase, as well (98).

63

How the inhibition is initiated - possible mechanisms

Regarding the inhibition caused by the C3a-derived peptides, we suggest two

mechanisms. One possibility is that binding of the peptide to the β-chain do not interfere with

the translocation of the aggregated FcεRIs into the lipid raft, but enhances the activation of the

Cbl ubiquitin ligase. On the other hand it is also likely that the peptide somehow inhibits the

association of the cross-linked receptors with the lipid rafts, therefore the activation of the Lyn

kinases is inhibited. This then results in the decreased phosphorylation of Lyn and the β-

subunit. Since the activity of the Lyn src kinase, and therefore the phosphorylation of the

receptor subunits is highly dependent on the lipid raft environment (58,128), we plan to

investigate whether the peptide affects the translocation of the antigen-clustered FcεRI into

the lipid raft. At present we can firmly state that the C3a-derived peptides interfere with the

FcεRI coupling network at its very earliest step, even prior to the important amplification

processes.

Further steps of the signaling cascade

PI3K kinase has a crucial role in MC degranulation (105) and is a key regulatory molecule

in the FcεRI-coupling signaling. The products of active PI3K are PI(3,4)P2 and PI(3,4,5)P3,

which can recruit several pleckstrin homology (PH) domain-containing molecules to the

plasma membrane (such as Btk, PLCγ and Akt), where they may become activated (47,49).

Our result shows that in MCs pre-treated with the complement-derived peptides (C3a7, C3a9)

the phosphorylation of the p85 regulatory domain of PI3K is inhibited (98). This inhibition

not only results in decreased degranulation but also a decreased level of PI(3,4,5)P3. As a

consequence, less PH-domain containing signaling molecules are recruited to the plasma

membrane causing the decrease of the MCs’ response.

We also investigated the antigen-induced transient rise in [Ca2+]i-level in MCs and found

that it is inhibitid by C3a9 (98).

It is well recognized that MCs have an important role in the initiation of inflammatory

response by the secretion of several mediators, including cytokines (IL-1-6, IL-9-14, IL-16,

IL-18, TNF-α, GM-CSF etc.), chemokines (IL-8, MIP-1, MIP-3 etc) and lipid mediators

(14,111). The de novo synthesis of cytokines is regulated by a variety of transcription factors

which are activated by different coupling pathways, including the MAPK-pathway (129). It is

known that FcεRI-clustering activates three members of the MAPK family, namely ERK1/2

(107), p38 (109) and JNK (108). Furthermore it has been described that each MAPK activates

64

different transcription factors, while one gene can be regulated by distinct transcription factors

which are activated by different MAPKs (129,130). The FcεRI-mediated activation of

ERK1/2 leads to the production of TNF-α and lipid mediators (111,129), while the activation

of p38 regulates the expression of IL-4 and IL-13 (131,132) and affects only marginally

TNF-α production (111). Here we show that C3a9 has an inhibitory effect on the

phosphorylation of ERK1/2 and p38. It is supposed that the main signaling route of ERK is

the Ras-Raf-MEK pathway (107), however several further activators of Raf are known, such

as Src PTK, PKC and Akt (133). Furthermore, the FcεRI-mediated ERK activation seems to

be independent of PI3K activation and the specific inhibitor of PI3K have only marginal

effect on ERK activation (134). Graham et al. suggest that ERK can be activated by a Ras-

independent way, as inhibitors of Ras do not affect FcεRI-induced ERK activation (135). It

has been reported that the growth factor-mediated MEK activation is Raf-independent and

also MEK can be activated by PKA in a Raf-independent-way, however this alternative

activator is still unknown (136,137). Since we found that C3a9 does not inhibit the activation

of Raf, we assume that the peptide inhibits the activation of ERK by an alternative route, not

via the Ras/Raf/MEK pathway. However, this alternative pathway still needs to be pursued.

Cytokine production by MCs

We have earlier shown that the complement-derived peptides, C3a7 and C3a9, inhibit

FcεRI-clustering induced degranulation of both the rat cell-line, RBL-2H3 and the bone

marrow-derived mast cells (BMMCs) (98). As these cells are known to regulate the immune

response by the production of a wide range of cytokines (1,2), next we investigated the effect

of the peptides on the late phase activation of MCs. We found that the secretion of TNF-α by

RBL-2H3 cells is significantly reduced upon peptide-treatment (98). It was highly important

to see, whether cells of physiological origin behave similarly. That is why we investigating

the effect of C3a9, the more potent peptide on the secretion of inflammatory cytokines by

BMMCs. We found that the production of the inflammatory cytokines (IL-6 and TNF-α) is

significantly inhibited in the presence of C3a9. These results demonstrate that the C3a-derived

peptides inhibit the late phase of MC activation, as well.

65

Actin is not involved in the inhibitory process

Actin cytoskeleton is known as a regulator of the distribution of membrane regions and

proteins upon receptor ligation in lymphocytes (81). In MCs F-actin regulates and stabilizes

protein distribution and lipid segregation in the membrane (82). By contrast, the actin

cytoskeleton has also been shown to play a negative regulatory role during MC activation, in

the process of the Lyn-FcεRI association and the immediate and late phase response, as well

(84). Therefore we assumed that the actin cytoskeleton might play role in the inhibitory

mechanism exerted by the complement-derived peptide. We found that the C3a9-mediated

inhibition of MCs is unaffected by the presence of Cytochalasin D, which disturbes F-actin

polymerization (113). Here we show that C3a9 pretreatment enhances the endocytosis of

aggregated receptors, a process known to be dependent on actin cytoskeleton. This finding

might be supported by studies demonstrating that F-actin has no obligatory role in receptor

internalization, but simply facilitates this process. Moreover, it has also been shown in several

cell types that Cytochalasin D treatment does not affect endocytosis (138,139).

Inhibition of anaphylaxis in vivo

As we have demonstrated, the C3a-derived peptides inhibit both the intermediate and

the late phase of MC activation in vitro (98), it was important to see whether C3a9 has any

effect in vivo, as well. We used a mouse model, the IgE-dependent passive systemic

anaphylaxis (PSA) for the test. In this reaction histamine has a crucial role in the

manifestation of the characteristic symptoms, such as changes in respiration pattern, and

hypothermia (140). It has been reported that the systemic histamine release is dependent on

both mast cell activation and FcεRI signaling (114). We have found that the FcεRI-mediated

systemic histamine release significantly decreased in the C3a9-treated mice. This result

clearly shows that C3a9 is an effective inhibitor of MC activation in vivo, as well.

Human system

Since our final goal is to develop anti-allergic substances for human therapeutic use, it

was also important to see how the complement-derived peptides effect the function of human

cells. The problem of in vitro studies in human MC research is the difficulty of isolation.

Therefore we used human basophil granulocytes instead of MCs. However the basophil

granulocytes are different from MCs in several respects, both cell types express the tetrameric

FcεRI and upon activation release several mediators (15). Basophil granulocytes are also

66

known to have a role in the pathogenesis of certain types of allergy (141-143) and these cells

are frequently used to assess the effectiveness of anti-allergic drugs (144,145). Our results

clearly show that the complement-derived peptides inhibit the activation of human basophil

granulocytes, as well, therefore we assume that these molecules can be potential therapeutic

agents to be used in allergic diseases. We compared the effect of C3a9 to other commercially

available anti-allergic drugs, such as chromolyn sodium, hydrocortison and dexamethason.

We consider it very important that C3a9 is a more potent inhibitor of basophil- activation than

the any other anti-allergic agents tested in our experimental system.

Dimers and dimerization of the peptides

We aimed to develope more effective and stable molecules based on the sequence of the

most effective inhibitory peptide C3a9 (DCCNYITR). In our attempt to map the active center,

first we checked the role of the two cysteins. We have synthetized dimers with intermolecular

disulphid-links and found that these peptides are very active in vitro (degranulation assay) and

in vivo (PSA), moreover they are potent inhibitors of the activation of human basophil

granulocytes, as well. These results strongly suggest that C3a9 exerts its effect as a dimer, and

presently we can not exclude the possibility that a „spontaneous dimerization” occurs when

we apply the monomeric peptides. This assumption is further supported by our finding that if

the formation of the disulphid-link was prevented, the inhibitory effect of the complement-

derived peptide was abolished. Taken together, we can say that the C3a-derived peptide C3a9

could be an effective inhibitory molecule of MC activation in its dimeric form and either this

molecule or its peptidomimetic derivative can be a lead for the development of a new anti-

allergic agent.

The possible phisiological role of MC inhibition by C3a

Regarding the phisiological aspect, based on our results the role of C3a in the immediate

hypersensitivity reaction can be viewed in a novel way. This peptide which is released upon

activation of the complement cascade is known to activate serosal type MCs and induce an

inflammatory response. Our results however, have demonstrated that C3a has another, earlier

not identified role, as well: it regulates MC activation negatively by binding to the FcεRI-β-

chain. It has been shown that several allergens, particularly inhaled ones are able to activate

the complement system, moreover trypsin-like enzymes expressed by various cell types (such

as macrophages, MCs, lymphocytes) can directly cleave C3, the third component of

complement. Freshly generated active C3a peptides activate serosal type MCs in their vicinity

67

through C3a receptors immediately. In this phase of the reaction the inhibitory capacity of the

intact anaphylatoxic peptide is most probably overrided by the activation through the C3a

receptor. As it is known that the G-protein coupled C3a-receptor uses a different signaling

pathway than the FcεRI-coupling network, intact C3a most probably exerts an additive effect

(95). However, when the C-terminal arginin of C3a is cleaved off by carboxypeptidase B,

which happens in a short time, C3a-desArg is generated. This degraded molecule does not

contain the activator sequence any more, consequently it is unable to induce the release of of

allergic mediators from serosal type MCs. C3a-desArg has a longer half-life than C3a and,

still contains the inhibitory stretch, thus it is able to inhibit the IgE-mediated activation of both

types of MCs present in mucosal surfaces, around blood vessels and nerves (146) (Fig.31.). In

conclusion, we have shown that C3a and its derivatives inhibit the MCs’ immediate and also

the late phase response by interfering with the early steps of the FcεRI coupling network by

binding to the β-chain of FcεRI.

Figure 31.: Possible interaction of C3a with serosal and mucosal type MCs.

Intact C3a can activate serosal type MCs through the C3a receptor. The activity of C3a is lost

by cleaving off the Arg from the C-terminal region. Both C3a and its inactive form (C3a-

desArg) may bind to the β-chain of FcεRI and inhibit the IgE-mediated activation both

serosal and mucosal type MCs.

68

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Summary MCs and basophil leukocytes are known as the main effector cells of allergic disorders.

These cells can be activated by various stimuli, including antigen/IgE or complement factors.

We have earlier described that the complement-derived anaphylatoxic peptide C3a, which can

activate serosal type mast cells, inhibit the IgE-mediated activation of the mucosal type cells.

Based on the sequence of C3a two inhibitory peptides were synthetized: C3a7

(CCNYITELR), which is the natural sequence of C3a, and C3a9 (DCCNYITR), its derivative.

Here we have shown that C3a9, the more potent complement-derived peptide binds to the

FcεRI-β-chain - similarly to C3a -, and remains bound even after receptor clustering. We have

shown that pretreatment of MCs with C3a9 enhances the internalization of FcεRI after antigen

challenge, right in the time-interval where the signaling cascade is initiated. Next we

investigated the association between the FcεRI-β-chain and two src kinases (Lyn and Fyn)

which are crucial for the initiation of receptor coupling network. We found that both the

association and phosphorylation of the β subunit and the Lyn src kinase are decreased in the

presence of peptide C3a9. We have shown that the C3a-derived peptides inhibit the general

phosphorylation pattern of intracellular proteins and the antigen-induced [Ca2+]i elevation, as

well. These results suggests that C3a9 inhibits the receptor proximal events, i.e. the earliest

steps of FcεRI-coupling network.

Mast cells release several cytokines that regulate the immune response and play a role in

the pathomechanism of allergy. The expression of cytokine genes are regulated by the

MAPK-pathway. Here we have described that C3a9 inhibits the activation of MAPKs

(ERK1/2 and p38) and it also decreases the production of inflammatory cytokines (TNF-α

and IL-6) by MCs.

We have shown by an in vivo model, that administration of C3a9 and its dimerized

derivatives into mice decrease the FcεRI-mediated histamine release in the passive systemic

anaphylaxis (PSA). It is also demonstrated, that the same peptides exert an inhibitory effect

on the activation of human basophil granulocytes.

In conclusion, we propose that the C3a-derived peptides inhibit MC response by

interfering with the early steps of the FcεRI coupling network, by binding to the β-chain of

FcεRI. Thus these peptides may serve as a basis for the further development of anti-allergic

substances.

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Összefoglalás A hízósejtek és a bazofil granulociták az allergiás reakciók fő effektor sejtjei. Ezek a

sejtek számos különböző stimulusra képesek válaszolni; így például az antigén/IgE

kölcsönhatást követően illetve különböző komplement-eredetű faktorokra. Munkacsoportunk

korábban leírta, hogy a C3a anafilatoxikus peptid, ami a szeróza típusú hízósejteket aktiválja a

C3a receptoron keresztül, gátolja a mukóza típusú hízósejtek IgE-mediált aktívációját.

Feltérképeztük, hogy a C3a molekulának mely része felelős ezért a gátlásért, és az azonosított

szakasz aminosav-szekvenciája alapján két peptidet szintetizáltattunk: a C3a7-et

(CCNYITELR), amely a C3a természetes szekvenciája, illetve C3a9-et (DCCNYITR), ami

ennek módosított származéka.

A dolgozatban bizonyítottuk, hogy a C3a9, ami a leghatékonyabb gátló peptidnek

bizonyult, a C3a-hoz hasonlóan az FcεRI β-láncához kötődik mind nyugvó, mind antigénnel

stimulált sejteken. A C3a9-es peptiddel kezelt sejtek esetében az antigénnel keresztkötött

FcεRI internalizációja fokozódott éppen abban az idő-intervallumban, amikor a jelátviteli utak

elindulnak. Kimutattuk, hogy a receptor és két src kináz, a Lyn és a Fyn, asszociációja - mely

elengedhetetlen kezdeti lépése az FcεRI-közvetített jelátviteli utaknak -, csökkent a peptid

jelenlétében, ugyanúgy, mint a receptor β alegysége és a Lyn kináz tirozinon való

foszforilációja. Leírtuk továbbá, hogy a C3a eredetű peptidek gátolják számos intracelluláris

fehérje foszforilációját és az antigén indukálta [Ca2+]i-szint növekedést is. Ezen eredmények

azt bizonyítják, hogy a C3a eredetű peptidek az FcεRI-közvetített jelátviteli folyamatok

legkorábbi lépéseit gátolják azáltal, hogy a receptor β-láncához kapcsolódnak.

A hízósejtek számos citokint is termelnek, melyek szerepet játszanak az immunválasz

szabályozásában és az allergiás folyamatokban. A citokin-gének expressziójának a

szabályozásában résztvesznek a MAP kinázok is. Kimutattuk, hogy a C3a9-es peptid gátolja

az ERK1/2 és p38 kinázok foszforilációját, és a TNF-α és IL-6 citokinek termelését is.

Egér in vivo modell alkalmazásával kimutattuk, hogy a hízósejtek FcεRI-n keresztüli

aktívációjához kapcsolható vér hisztamin-szint növekedést is gátolják a komplement eredetű

peptidek. Továbbá azt is bizonyítottuk, hogy a humán bazofil granulociták FcεRI-en

keresztüli stimulációját is szignifikánsan csökken a C3-eredetű peptidek jelenlétében.

Eredményeink alapján tehát azt mondhatjuk, hogy a C3a-eredetű peptidek a hízósejtek

FcεRI-dependens válaszát már a legkorábbi aktivációs lépések blokkolásával érik el, és

alkalmasak anti-allergikum fejlesztésére..

81

Acknowledgements

First of all I would like to thank my supervisor Prof. Erdei Anna that she allowed me to

work at the Department of Immunology in the Eötvös Loránd University and all the support

and scientific help during my research project. I am very thankful for the lot of technical help

to Zsuzsa Szabó, Krisztina Kerekes, Vera Barbai and Márton Andrásfalvy. I thank all my

colleagues at the Departement of Immunology for the excellent working environment.

I have to thank Prof. Israel Pecht for the possibility to work at the Department of

Immunology in the Weizmann Institute, Rehovot, Israel, and for all the support I got from

him. I would like to thank Alina Barbu and Jakub Abramson for their help and kindness to

have a nice and useful time in Israel.

82

List of publications Publications connected to the thesis

Márton Andrásfalvy* et Hajna Péterfy*, Gábor Tóth, János Matkó, Jakub Abramson,

Krisztina Kerekes, György Vámosi, Israel Pecht, Anna Erdei

The β subunit of the type I Fcε receptor is a target for peptides inhibiting IgE-mediated

secretory response of mast cells

The Journal of Immunology, 2005, 175: 2801-2806 IF: 6.486

* contributed equally to this work

Anna Erdei, Márton Andrásfalvy, Hajna Péterfy, Gábor Tóth, Israel Pecht

Regulation of mast cell activation by complement-derived peptides

Immunology Letters, 2004, 92: 39-42 IF: 2.136

Hajna Péterfy, Gábor Tóth, Israel Pecht, Anna Erdei

C3a derived peptide binds to the type I Fcε receptor and inhibits proximal coupling processes

and cytokine secretion by mast cells

Submitted to J.Immunol., 2007

Other publication

Kovacs Z, Kekesi KA, Szilagyi N, Abraham I, Szekacs D, Kiraly N, Papp E, Csaszar I, Szego

E, Barabas K, Peterfy H, Erdei A, Bartfai T, Juhasz G.

Facilitation of spike-wave discharge activity by lipopolysaccharides in Wistar Albino

Glaxo/Rijswijk rats.

Neuroscience, 2006, 140(2): 731-742

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Published abstracts

Márton Andrásfalvy, Gábor Tóth, Hajna Péterfy, György Vámosi, János Matkó, Israel Pecht,

Anna Erdei

Inhibition of FcεRI-clustering induced activation of mucosal type mast cells by C3a-derived

peptides

Immunology Letters, 2003, Vol.: 87

Hajna Péterfy, Márton Andrásfalvy, Gábor Tóth, János Matkó, Jakub Abramson, György

Vámosi, Israel Pecht, Anna Erdei

The β subunit of the type I Fcε receptor is a target for vomplement-derived peptides inhibiting

IgE-mediated secretory response of mast cells

The FEBS Journal, 2005, Vol.: 272

Oral presentations

Hajna Péterfy, Gábor Tóth, Israel Pecht, Anna Erdei

C3a drived peptide binds to FcεRI and inhibits proximal coupling processes and cytokine

secretion by mast cells

14th Signals and Signal Processing in the Immune System, Balatonöszöd, Hungary - 2007

Posters

Hajna Péterfy, Gábor Tóth, Israel Pecht, Anna Erdei

Changes in membrane proximal events and cytokine production of mast cells stimulated by

antigen in the presence of inhibitory peptide C3a9

36th. Conference of Hungarian Society for Immunology, Hajdúszoboszló, Hungary - 2007

Hajna Péterfy, Vera Barbai, Krisztina Kerekes, Gábor Tóth, Israel Pecht, Anna Erdei

The effect of complement-derived inhibitory peptides on mast cell signaling

16th European Congress of Immunology, Paris, France - 2006

84

Hajna Péterfy, Vera Barbai, Krisztina Kerekes, Gábor Tóth, Israel Pecht, Anna Erdei

The complement-derived peptide inhibits the MAPK-pathway upon FcεRI-cross-linking in

mast cells

35th Conference of Hungarian Society for Immunology, Sopron, Hungary – 2005

Hajna Péterfy, Vera Barbai, Alina Barbu, Gábor Tóth, Israel Pecht, Anna Erdei

Complement-derived peptide has selective affact on the MAPK-pathway in mast cells

13th Signals and Signal Processing in the Immune System, Balatonöszöd, Hungary – 2005

Hajna Péterfy, Bernadett Balla, Anna Erdei

In vitro study of the involvement of CD21 in the maturation of bone marrow derived mast

cells

The Batsheva de Rothschild International Workshop on Mast Cell Signaling and Function in

Health and Disease, Eilat, Israel - 2005

85