Post on 01-Jun-2020
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
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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
83
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