fulltext01

Linköping University Medical Dissertations
No.996
Pro- and Anti-inflammatory Regulation
of β2 Integrin Signalling
in Human Neutrophils
Veronika Patcha Brodin
Division of Cell Biology
Department of Biomedicine and Surgery
Faculty of Health Sciences, Linköping University
SE-581 85 Linköping
SWEDEN
Linköping 2007
Cover image: Fluorescence image of differentiated HL60 cells phagocytosing yeast. Shown in
the image; F-actin depicted in red, and yeast in grey.
© Veronika Brodin
All rights reserved
ISSN: 0345-0082
ISBN: 978-91-85715-22-0
The published articles are reprinted with the permission from the publishers
Printed by LiU-Tryck, Linköping, Sweden 2007
To Greger, Ella, and Alexander
…and all the curious people out there
ABSTRACT
The body is under constant attack from pathogens trying to slip by our immune defence. If the
barrier is breached, invading pathogens enter the tissues and cause inflammation. During this
process neutrophils, constituting the first line of defence, leave the bloodstream and seek out
and kill the invading pathogens. The mechanisms leading to activation of receptors on
neutrophils must be closely orchestrated. Pro- and anti-inflammatory substances can influence
the outcome of the inflammation process by affecting the involved players. If not well
balanced, inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, can be the
outcome.
The aim of this thesis was to elucidate the effect of pro- (fMLP, Leukotriene B4, and
Interleukin-8) and anti- (lipoxins, aspirin and statins) inflammatory substances on the β2
integrins, mediating adhesion of neutrophils both under “normal” conditions and during
coronary artery disease. More specifically, the effect of these substances on the β2 integrins
were studied in regard to: i) the activity (i.e. affinity and avidity) of β2 integrins, ii) the
signalling capacity of β2 integrins (i.e. detected as release of arachidonic acid, and the
production of reactive oxygen species, and iii) the signal transduction mediated by the β2
integrins (i.e. phosphorylation of Pyk2).
The pro-inflammatory substances belong to the family of chemoattractants that induces
transmigration and chemotaxis. A hierarchy exists between the different family members; the
end-target
chemoattractants
(e.g.
fMLP)
being
more
potent
than
intermediary
chemoattractants (e.g. IL-8 and LTB4). It was found that intermediary chemoattractants
B
regulate β2 integrins by mainly affecting the avidity of β2 integrins. End-target
chemoattractants on the other hand, affected the β2 integrins by increasing the avidity and the
affinity, as well as their signalling capacity.
The anti-inflammatory substances used in this study were the exogenous aspirin and statins,
and the endogenous lipoxins. In the presence of aspirin, stable analogues of lipoxin (i.e. epilipoxins) are formed in a trans-cellular process. Lipoxin inhibited the signalling capacity of β2
2
integrins mediated by intermediary chemoattractants, as well as the signal transduction
induced by end-target chemoattractants. Moreover, the signalling capacity of β2 integrins in
neutrophils from patients suffering from coronary artery disease (CAD) was impaired.
Arachidonic acid, the precursor for both pro- and anti-inflammatory eicosanoid, induced an
increase in the β2 integrin activity (both affinity and avidity), but had no effect on the signal
transduction.
In conclusion, different “roles” were observed for end-target and intermediary
chemoattractants in the regulation of β2 integrins. The inhibitory effects of the antiinflammatory lipoxins support earlier studies suggesting that these agents function as “stop
signals” in inflammation. This is also confirmed by our findings in CAD patients, who have
elevated levels of epi-lipoxins due to aspirin treatment. Moreover, Pyk2 was identified as a
possible target for the inhibitory effect of anti-inflammatory drugs.
3
PREFACE
This thesis is based on the following papers, referred to by their Roman numerals (I-V):
I)
Patcha V, Wigren J, Winberg ME, Rasmusson B, Li J, and Särndahl E:
Differential inside-out activation of β2-integrins by leukotriene B4 and fMLP in
human neutrophils. Exp. Cell Res. 300: 308-318, 2004.
II)
Patcha Brodin V and Särndahl E: Lipoxin A4 inhibits the fMet-Leu-Pheinduced, but not β2 integrin-induced activation of the non-receptor tyrosine
kinase Pyk2 in human leukemia cells (HL-60). Manuscript 2007, submitted
III)
Patcha Brodin V and Särndahl E: Inside-out regulated, β2 integrin-induced
release of arachidonic acid in Human Leukemia 60 cells. Manuscript 2007,
submitted
IV)
Lerm M, Patcha Brodin V, Ruishalme I, Stendahl O, and Särndahl E:
Inactivation of Cdc42 is necessary for depolymerization of phagosomal F-actin
and subsequent phagosomal maturation. Accepted for publication in J. Immunol.
V)
Särndahl E, Bergström I, Patcha Brodin V, Nijm J, Lundqvist-Setterud H, and
Jonasson L: Neutrophil activation status in stable coronary artery disease.
Manuscript 2007, submitted
4
5
CONTENTS
ABSTRACT........................................................................................................................................................... 2
PREFACE.............................................................................................................................................................. 4
CONTENTS........................................................................................................................................................... 6
ABBREVIATIONS ............................................................................................................................................... 8
INTRODUCTION............................................................................................................................................... 12
ADHESION ........................................................................................................................................................ 12
THE ACTIN CYTOSKELETON ............................................................................................................................. 13
INTEGRINS ........................................................................................................................................................ 14
Integrin activation: affinity, avidity, and valency........................................................................................ 16
β2 integrin ligation ..................................................................................................................................... 18
INTEGRINS, PARTNERS IN SICKNESS AND IN HEALTH ...................................................................................... 20
INTEGRINS, MATRIX ADHESIONS AND ASSOCIATED PROTEINS ........................................................................ 21
Cytoskeletal proteins associated with adhesion complexes......................................................................... 21
Proteins involved in mediating signal transduction induced by integrins................................................... 23
CELL MOTILITY ................................................................................................................................................. 25
REGULATION OF ADHESION SIGNALLING BY LIPID METABOLITES ................................................................... 26
Phosholipase A2 .......................................................................................................................................... 26
Arachidonic acid and pro-inflammatory eicosanoids.................................................................................. 27
Anti-inflammatory lipoxins .......................................................................................................................... 28
PHAGOCYTOSIS................................................................................................................................................ 29
The phagocytic process ............................................................................................................................... 29
Degranulation.............................................................................................................................................. 30
AIMS OF THE INVESTIGATION................................................................................................................... 31
METHODS .......................................................................................................................................................... 33
NEUTROPHILS .................................................................................................................................................. 33
[3H]ARACHIDONIC ACID-LABELLING AND RELEASE ........................................................................................ 34
CELL STIMULATION .......................................................................................................................................... 34
Activation by chemotactic factors................................................................................................................ 34
Integrin ligation........................................................................................................................................... 34
FLOW CYTOMETRY ........................................................................................................................................... 35
Expression of β2-integrins.......................................................................................................................... 37
β2 integrin affinity ...................................................................................................................................... 37
Binding of soluble ICAM-1.......................................................................................................................... 38
FLUORESCENCE MICROSCOPY ........................................................................................................................ 38
SINGLE PARTICLE TRACKING .......................................................................................................................... 38
CONFOCAL SCANNING LIGHT MICROSCOPY................................................................................................... 39
IMAGE ANALYSIS .............................................................................................................................................. 40
Clustering of integrins ................................................................................................................................. 40
Granule distribution .................................................................................................................................... 40
INTRODUCTION OF RECOMBINANT PROTEINS INTO HUMAN NEUTROPHILS ..................................................... 40
HIV TAT PROTEINS .......................................................................................................................................... 41
TAT transduction ......................................................................................................................................... 41
IMMUNOPRECIPITATION AND WESTERN BLOT ................................................................................................. 42
PHAGOCYTOSIS AND PRODUCTION OF REACTIVE OXYGEN SPECIES .............................................................. 42
STATISTICAL ANALYSIS.................................................................................................................................... 42
RESULTS ............................................................................................................................................................ 44
THE EFFECT OF PRO- AND ANTI-INFLAMMATORY CHEMOATTRACTANTS ON THE ACTIVATION STATE OF THE
β2 INTEGRINS .................................................................................................................................................. 44
6
The activation state of β2 integrins on neutrophils from healthy donors, mediated by pro-inflammatory
chemoattractants ......................................................................................................................................... 44
The effect of anti-inflammatory substances on the activation state of β2 integrins.................................... 45
The activation state of β2 integrins on neutrophils from patients with stable CAD ................................... 46
THE EFFECT OF PRO- AND ANTI- INFLAMMATORY SUBSTANCES ON THE SIGNALLING CAPACITY OF β2
INTEGRINS ........................................................................................................................................................ 47
Signalling capacity, as measured by the release of arachidonic acid ......................................................... 47
The signalling capacity of β2 integrins, measured by production of reactive oxygen species ................... 50
THE EFFECT OF PRO- AND ANTI-INFLAMMATORY SUBSTANCES ON THE SIGNAL
TRANSDUCTION MEDIATED BY β2 INTEGRINS.................................................................................... 51
ADDITIONAL METHODS TO STUDY PROTEINS IMPORTANT FOR THE REGULATION OF β2 INTEGRINS ............. 54
TAT-protein transduction ............................................................................................................................ 55
Transduction of HL60 cells with TAT-Pyk2 ................................................................................................ 57
SUMMARY ......................................................................................................................................................... 58
CONCLUDING REMARKS AND FUTURE PERSPECTIVES.................................................................... 59
ACKNOWLEDGEMENTS................................................................................................................................ 60
REFERENCES.................................................................................................................................................... 65
7
ABBREVIATIONS
AA
ATL
BSA
CAD
COX
CR3
CRP
C3b
C3bi
DMSO
EDTA
EGTA
ECM
F-actin
FcR
FSC
FAK
FITC
fMLP
GAP
GDI
GDF
GDP
GEF
G-protein
GPCR
GTP
HA
HETE
HL60
HPETE
HRP
HSA
ICAMs
IgG
IL-8
LAD
LFA-1
LOX
LTs
arachidonic acid
aspirin-triggered lipoxins
bovine serum albumine
coronary artery disease
cyclooxygenase
complement receptor 3 (also known as; CD11b/CD18, β2-integrin,
Mac-1)
C-reactive peptide
fragment of complement factor 3
inactivated C3b
dimethylsulphoxide
ethylenediaminetetraacetic acid
glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
extracellular matrix
filamentous actin
Fc-Receptor
forward scatter
focal adhesion kinase
fluorescein isothiocyanate
N-formyl-L-methionyl-L-leucyl-L-phenylalanine, (f-Met-Leu-Phe)
GTPase activating protein
guanine nucleotide dissociation inhibitor
GDI displacement factor
guanosine diphosphate
guanine nucleotide exchange factor
GTP-binding protein
G-protein-coupled receptors
guanosine triphosphate
hemagglutinine
hydroxyeicosatetraenoic acid
human leukemia 60
hydroperoxyeicosatetraenoic acid
horseradish peroxidase
human serum albumine
intracellular adhesion molecules
immunoglobulin G
interleukine 8
leukocyte adhesion deficiency
lymphocyte function-associated antigen 1 (αLβ2)
lipoxygenase
leukotrienes
8
LTB4
LX
LXa
LXA4
MAPK
MAPTA/AM
MFI
NADPH
PAF
PFA
PGs
PI-3 K
PKC
PLA2
cPLA2
sPLA2
PMA
PMB
PMN
PTKs
Pyk2
ROS
S.E.M.
SOD
SPT
SSC
Tat
TAT
TNF
leukotriene B4
lipoxin
lipoxin analogue
lipoxin A4
mitogen activated protein kinase
1,2-bis-5-methyl-amino-phenoxyl-ethane-N,N,N´-tetraacetoxymethyl acetate
mean fluorescence intensity
nicotinamide adenine dinucleotide phosphate
platelet activating factor
paraformaldehyde
prostaglandins
phospatidylinositol-3 Kinase
protein kinase C
phospholipase A2
cytosolic PLA2
secretory PLA2
phorbol 12-myristate acetate
polymyxin B
polymorphonuclear neutrophils
protein tyrosine kinases
proline-rich tyrosine kinase 2
reactive oxygen species
scanning electron microscopy
superoxide dismutase
single particle tracking
side scatter
transcriptional activator of transcription of HIV
transduction domain of HIV Tat (11 aa)
tumour necrosis factor
B
9
It was the darkest of times.
Evil forces thrived in the world. An enemy invaded the
land. But there was hope. The ”White Army” secretly prepared for war on the islands of the
Red River. Armed with powerful weapons, they formed the “first line of defence”. Brave
soldiers breached the Endothelial Wall in the quest to seek out the vile enemy. The climb in
the ECM-Mountains was steep and dangerous. The tracks were easily followed, the foul smell of
the invaders lingering in the air. Finally, the enemy camps were in reach. Equipped with deadly
toxins, the soldiers attacked. The battle went on for days. Darkness prevailed, but in the end
hope returned. Out-numbered, the enemy perished. The land slowly began to heal, and soon
no signs of invasion were seen.
Once again the inhabitants of the land lived in peace and harmony, knowing that somewhere
out there…the quiescent White Army... awaits the next attack...
The White Army
Enemy Camp
Red River
Pus-Swamps
ECM-Mountains
Endothelial Wall
10
11
INTRODUCTION
Adhesion
The ability to adhere is crucial for many cellular processes. The adhesive interactions are
usually transient, but can become static. During embryogenesis, organs are formed by static,
homotypic adhesion between cells. In fertilization, the sperm needs to bind to the egg cell for
new life to begin. During inflammation, white blood cells (e.g. neutrophils) adhere to, and
squeeze between the endothelial cells lining the blood vessels, and seek out invading
pathogens. At the site of infection, the white blood cells must adhere to the bacteria in order to
engulf them. Many different adhesion receptors are involved in these processes. Cadherins
form the static connections between cells. Selectins mediate the initial, loose attachment of
neutrophils to the endothelium. Integrins are involved in both firm adhesion to the
endothelium, adhesion to extracellular matrix (ECM) during chemotaxis, and during
phagocytosis in the adhesion between cells and pathogens during phagocytosis (Fig. 1)
(reviewed in (1)).
Rolling
Firm adhesion
Diapedesis Chemotaxis
Phagocytosis
Endothelium
+
Selectins
Carbohydrates
ICAMs
Integrins (CR)
GPCR
C3bi
Antibody
FcR
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + +
+
+
+
12
+
+
+
+
+
+ +
+ +
+
+ +
+
+
+
+
+
Figure 1. The inflammatory process. During inflammation, P- and E-selectins are
upregulated on the activated endothelium. L-selectins on neutrophils bind loosely to
ligands on the endothelial cells, and rolling of the cells on the blood vessel is initiated.
Integrins
are
upregulated
on
neutrophils
upon
stimulation
with
chemokines/chemoattractants or by selectin-mediated rolling (1). Integrins adhere firmly
to the endothelium upon binding to ICAMs (reviewed in (2)). After locating “gaps”
between endothelial cells, neutrophils, following a gradient of chemoattractants (+),
squeeze through the endothelial layer (diapedesis) and migrate towards the site of
infection (chemotaxis). Pathogens opsonized with complement factors (C3b/C3bi) or
antibodies are engulfed via complement receptor (CR)-mediated or Fc-receptor (FcR)mediated phagocytosis. Reactive oxygen species and enzymes degrade and kill the
intracellular pathogens.
The actin cytoskeleton
Reorganization of the cytoskeleton is an essential process during adhesion, chemotaxis and
phagocytosis. Actin filaments form a three-dimensional network called the actin cytoskeleton,
which stabilizes the cell. The actin cytoskeleton is remodelled during cell migration, the
filamentous-actin (F-actin) being polymerized at the plus (barbed) end, and depolymerized
into globular-actin (G-actin) at the minus (pointed) end (Fig. 2). Polymerization of actin is
dependent of ATP and monovalent or divalent ions e.g. potassium (K+) and magnesium
(Mg2+) (reviewed in (3)). Cellular movement by so called “amoeba-like” migration requires
constant turnover of extracellular adhesive contacts, which are intracellularly connected to the
cytoskeleton. In order to move the cell body forward, a lamellipodium is extended at the
leading edge and attached to the substratum. Simultaneously, the adhesive connections in the
back of the cell are disconnected and the cell body is retracted (Fig. 2).
13
A
Leading edge
Retraction
Extention
ECM
B
G-actin
F-actin
-
i)
+
ii)
Figure 2. Cellular migration. A) During migration, the leading edge extends forward
and attaches to the extracellular matrix (ECM). In order to retract the cell body, the
adhesive connections must be severed at the trailing edge (uropod) of the cell. B) The
actin cytoskeleton is composed of monomeric, globular actin (G-actin) i) polymerizing
into actin filaments. Polymerization of filamentous actin (F-actin) ii) occurs at the plus
end, whereas depolymerization occurs at the minus end of the filaments.
Integrins
Integrins are transmembrane cell-surface receptors mediating adhesion to the ECM, and to
other cells or pathogens. Integrins consist of a α-subunit and a β-subunit, non-covalently
bound to each other. Today, 18 α-, and 8 β-subunits have been identified, which can be
combined into at least 24 different integrin receptors (4) (Fig. 3), all having a specific
function, as shown by the phenotypes of knockout mice (reviewed in (5)).
14
α1*
α2*
α3
α4
β1
β7
αE*
α5
α6
β4
α7
α8
α9
α10
β3
αL*
α11
β5
αv
β6
αIIb
β8
αM*
β2
(CD11a/CD18; LFA-1)
(CD11b/CD18; Mac-1,CR3, MO-1)
αX* (CD11c/CD18, CR4, gp 150/95)
αD* (CD11d/CD18)
Figure 3. The integrin superfamily. One α- together with one β-subunit constitutes
an integrin receptor. Also shown are the alternative designations for β2 integrins.
Integrins containing an I-domain are marked with an asterisk.
The integrin receptors consist of a large extracellular, ligand-binding domain, a
transmembrane domain, and a short cytoplasmic tail (Fig. 4). The CD11/CD18 integrins (β2
integrins), solely expressed on leukocytes, share the same β-subunit combined with four
different α-subunits (CD11a, -b, -c, -d). The CD11b/CD18 integrins are the most abundant β2
integrins on neutrophils.
In order to participate in cellular processes such as wound healing, cell differentiation,
immune responses, and cell migration, the integrins can not be constitutively active. The
ligand-binding affinity is closely regulated via conformational changes (Fig. 4) (5). Inactive
integrins have a bent conformation, folded at the genu, with the headpiece located 5 nm above
the membrane (6, 7). When activated, the unfolded integrins project 20 nm above the
membrane, and are accessible for ligand binding (8). It should be noted, however, that ligand
binding has been observed already for the “bent” conformation of αvβ3- integrins (9),
suggesting that the activation model does not apply to all integrins.
15
Closed, bent formation
Closed, extended formation
I-domain
Intrinsic
Ligand
Open, extended formation
Extrinsic
Ligand
βI-domain
(I-like domain)
β-propeller
Genu
Thigh domain
Hybrid domain
PSI domain
Calf
domains
α β
EGF repeats
α β
α
β
Signalling
Figure 4. The structure of integrins. The conformational changes in integrins
containing an I-domain. In a low affinity state, the α- and β-integrin tails are in a closed,
bent conformation. Upon priming (or ligand binding), the integrin receptor ”legs”
straighten up in a “switchblade”-like motion (10) and separate; a mechanism associated
with increased affinity. The separation of the legs, accompanied by conformational
changes allows for binding of cytoplasmic proteins and signalling. Adapted from (6, 1114).
Integrin activation: affinity, avidity, and valency
Besides the induction of conformational changes of integrins (affinity change), clustering of
the receptors on the plasma membrane, together with the association of integrins and the
cytoskeleton, are additional ways of activating the integrin receptors (avidity regulation).
However, if a conformational change of the integrins is the cause or the result of ligand
binding is under debate (15, 16).
Changes in affinity and avidity are brought about via an inside out mediated-mechanism,
induced by activation of other membrane receptors, e.g. receptors for growth factors,
cytokines and chemoattractants. Inside-out signalling, in which the cytoplasmic regions of β2
integrins are key players, is perceived as conformational changes in the extracellular region of
the integrins, mediated from the cytoplasmic side by the signal transduction induced by other
16
active receptors. The proteins affecting the inside-out signalling should posses the ability to
directly associate with the integrins (Table 1), or be involved in the signal transduction
resulting in the activation of integrin-associated molecules (reviewed in (17)). Changes in
affinity and avidity put the integrins in a different activation state, so called “primed state”,
giving the cells a more “readily activated” character.
The terminology surrounding integrin activation is vague. Recently, the term valency has
been introduced (14). Valency regulations include changes in the local density, or in the
ability of the receptor and ligand to move, which alter the possible number of adhesive bonds
that can be formed. Affinity regulations are explained as changes in integrin affinity due to
conformational changes. (14). The avidity is considered as the total strength of cellular
adhesive interactions resulting from the affinity, valency, and the total number of formed
bonds (18). In this thesis, the term valency is not applied, instead affinity and avidity will be
used (Fig. 5).
17
Affinity regulation
Avidity regulations
Upregulation
and
lateral mobility
Clustering
Conformational
change
Connection
to cytoskeleton
Cytoskeleton
Avidity regulation
β2 integrins
Figure 5. Schematic illustration of integrin activation regulated by affinity
and avidity changes. Upon activation, the number of integrin receptors is increased by
upregulation from intracellular stores. When the cytoskeletal connections are broken,
integrin receptors move laterally on the plasma membrane and form clusters, thereby
accumulating the number of low-affinity binding integrins in one area, resulting in
increased binding/adhesion. The ligand binding of integrins can also be strengthened by
altering the connection between receptors and the cytoskeleton. Affinity is increased by a
conformational change in the integrin receptor, resulting in augmented ligand binding.
β2 integrin ligation
Besides being adhesion receptors, integrins cross-talk with other receptors on the plasma
membrane, and transmit signals into the cell interior. The extracellular domain of β2 integrins
binds to the ECM, or to counter receptors on neighbouring cells. The cytoplasmic domain
interacts with cytoskeletal proteins, forming a physical link between ECM and the
cytoskeleton, thereby regulating the signalling capacity of the receptor. Ligation of the
receptor induces a signal that propagates within the cell, a process called outside-in signalling.
For example, besides inducing the activation of adhesion kinases localized to focal
complexes, ligation of integrins can activate mitogen activated protein kinase (MAPK), GTPbinding proteins (G-proteins), polymerization of F-actin, and induce release of Ca2+.
18
ICAM-1, was one of the first molecules to be identified as a ligand for leukocyte integrins
(19). ICAMs belong to the immunoglobulin superfamily consisting of five members: ICAM
1-5 (reviewed in (20, 21)). In addition to ICAMs, β2 integrins bind a vast number of
molecules including; fibrinogen, collagen I, and C3bi. In T-cells, the binding of soluble
ICAM (sICAM), corresponding to increased integrin affinity, is induced by divalent ions such
as Mg2+ and Manganese (Mn2+) (16, 22), (23). The integrin receptors contain multiple binding
sites for calcium (Ca2+) in their extracellular domains, and Ca2+ is another ion regulating
integrin activation/ligand binding. Surprisingly, Ca2+ has a dual role in both inducing
adhesion, by integrin clustering (24), and inhibiting adhesion. Actually, Ca2+ has to be
removed in order for Mg2+ to induce changes in affinity (23).
Table 1. A summary of proteins interacting with integrin cytoplasmic domains.
Adapted from (25).
Protein
Integrin tail
References
Filamin
β1A, β2, β7
(26, 27)
Talin
β1-5, αIIb, (β7)
(26, 28)
α-actinin
β2, β1
(29)
Radixin
β2
(30)
CIB
αIIb
(31)
Calreticulin
α
(32)
FAK (p125)
β
(33)
ILK (p59)
β
(34)
Pyk2
β2
(35)
Paxillin
β1, β2, β3, α4, α9
(33, 36, 37)
Cytohesin-1, and -3
β2
(38)
β3-Endonexin
β3
(39)
ICAP-1
β1
(40, 41)
Rack1
β1, β2, β3
(42)
Eps8
β1a
(43)
PI3 Kinase
β1
(44)
IRS-1
αvβ3
(45)
Phospholipase Cγ
β1
(46)
14-3-3 β
β1
(47)
Annexin-V
β5
(48)
19
Integrins, partners in sickness and in health
Leukocytes are key players in inflammation and thereby also potential targets for antiinflammatory drugs. Normally, non-adherent leukocytes circulate in the blood, become
activated, and transmigrate through the endothelium into the tissues. Unregulated
accumulation of leukocytes in target organs or tissues may result in diseases such as asthma,
rheumatoid arthritis, atherosclerosis, multiple sclerosis and Crohn’s disease. Moreover, the
elevated number of neutrophils in circulation increases the risk of developing cardiovascular
disease. Data from patients with leukocyte adhesion deficiency (LAD) and integrin
knockouts, confirm the central role of integrin signalling during inflammation. LAD-I is an
inherited immunodeficiency in which the expression of β2 integrins is diminished or lost (49).
LAD patients suffer from impaired inflammatory responses, defects in T cell proliferation,
and skin infections (reviewed in (5)). Other LAD deficiencies have been identified, where the
expression of β2 integrins is normal, but the function is impaired, probably due to defective
“inside-out signalling” (50, 51). In the last decade, neutrophils have been identified as active
participants in the inflammatory process of atherosclerosis, which in turn is a major risk factor
in the development of coronary artery disease (CAD).
20
Integrins, matrix adhesions and associated proteins
Integrins and their binding partners (summarized in Table 1) are organized in multi-protein
complexes termed “matrix adhesions”. They are highly dynamic and difficult to study as
isolated organelles. The matrix adhesions include focal complexes, focal adhesions (also
designated focal contacts), fibrillar adhesions and podosomes (52). Focal complexes are small
(100 nm), dot-like structures usually concentrated in membrane protrusions of migrating cells.
Focal complexes can mature into larger focal adhesions (1 μm) found mainly in resting cells
or in areas with low motility (52). Fibrillar adhesions are formed in cells adhering to
fibronectin via α5β1, and do not contain vinculin or paxillin (53). Podosomes are formed in
osteoclasts or cells of hematopoietic origin e.g. neutrophils (54-56).
Cytoskeletal proteins associated with adhesion complexes
Talin was the first protein identified as a cytoplasmic binding partner to integrins (28). Talin
is a cytoskeletal protein that participates in the activation of integrins, formation of adhesion
matrices, in connecting integrin receptors to the cytoskeleton (reviewed in (57)), and in β2
mediated phagocytosis (58). Talin binds with high affinity to the cytoplasmic tails of β
integrins via the N-terminal, FERM (i.e. protein 4.1, ezrin, radixin, moiesin) domain (26), and
to actin via the C-terminal domain. Recently, an alternative splicing product of talin, talin2,
has been identified (59). The function of talin2 remains to be elucidated. In neutrophils, the
connection between CD11a/CD18 and talin is disrupted upon activation, following an
association of the integrin receptors with α-actinin (60), the cytoskeletal protein that crosslink
F-actin. Paxillin is another adaptor protein important for the assembly and functions of matrix
adhesions. Paxillin, together with talin, is one of the first proteins recruited to focal contacts
(61).
Rho GTPases and cytoskeletal rearrangements
The regulation of the cytoskeletal rearrangements induced by chemoattractants is mediated by
the family of small GTPases. In neutrophils, research has mainly focused on the members
Rho, Rac, and Cdc42, which become transiently activated by N-formyl-L-methionyl-L-leucylL-phenylalanine (fMLP) (62-65). The transition of the GTPases between inactive, GDPbound and active, GTP-bound state is regulated by guanine nucleotide exchange factors
21
(GEFs), and GTPase activating proteins (GAPs) (reviewed in (52)). GEFs catalyze the
exchange of GDP for GTP during activation of the GTPases, and GAPs stimulate the low
intrinsic hydrolytic activity (Fig. 6). The activation of GTPases is further regulated by guanine
nucleotide dissociation inhibitors (GDIs) which prevent exchange of GDP to GTP, and hence
must be dissociated from the GTPases during activation (66).
GEF
Active
Rho GTPase
Inactive
Rho GTPase
P
GD
GAP
G
TP
I
GD
GDF
GDI
Figure 6. The activation model of Rho GTPases. The transition between
active/inactive state is modulated by guanine nucleotide exchange factors (GEFs),
GTPase activating proteins (GAPs), and guanine nucleotide dissociation inhibitors
(GDIs). The dissociation of RhoGDI and RhoGTPase is mediated by GDI displacement
factors (GDFs).
Post-translational lipid modifications at the C-terminal part target the GTPases to different
membranes/compartments (67), thereby exerting different effects on the cytoskeletal
rearrangements. In neutrophils, Rac is located in the front of the cells, mediating the
formation of leading edge and F-actin assembly (68, 69). The primary role of Rac is to induce
protrusive force during cell motility. Integrin ligation induces the translocation of inactive Rac
and Cdc42 from the cytoplasm to the membrane, and mediates the dissociation of RacGDI
(70). β2 integrin-ligation activates Rac and Cdc42 in neutrophils (71), and both Rac and
Cdc42 are translocated to the plasma membrane in an integrin-dependent manner (70). Cdc42
localizes to the leading edge of chemoattractant-stimulated neutrophils, and becomes
activated by p21-activated kinase (PAK) and the RacGEF, PIXα. Rho regulates myosin IImediated retraction of the tail in monocytes and neutrophils through its effector Rho kinase
22
(ROCK) (72, 73). Rho-H has been proposed to negatively regulate CD11a/CD18 avidity (74).
Rac and Rho are concentrated in lipid rafts (75), (76), and Rac co-localizes strongly with
GM1 (77, 78).
Rap1 and RAPL
Recently Rap1, a small GTPase belonging to the family of Ras-like GTPases has emerged as
an important regulator of integrin-mediated adhesion (79). Activated Rap1 induces increased
affinity and clustering of CD11a/CD18 in lymphocytes (80). Rap1 also regulates the
interaction between talin and integrins (81). In macrophages, Rap1 regulates complementmediated phagocytosis (82). Using yeast-two-hybrid screening, the Rap1-binding proteins
RAPL and Riam (Rap1-interacting adaptor molecule) have been identified (83-85). RAPL is
highly expressed in lymphocytes, and associates with the GTP-bound Rap1. Both RAPL and
Riam contain Ras-association domains but their function needs to be elucidated.
Proteins involved in mediating signal transduction induced by integrins
The protein tyrosine kinases
Plasma membrane receptors lacking intrinsic protein tyrosine kinase activities, i.e. integrins,
recruit non-receptor tyrosine kinases in order to mediate signals (86-92). The focal adhesion
kinase (FAK) belongs to the family of non-receptor tyrosine kinases and plays a central role
in integrin-mediated signalling (93). The FERM domain, located at the N-terminus of FAK,
binds β1 integrin cytoplasmic tails. At the C-terminus, FAK contains a focal adhesion
targeting (FAT) domain functioning as binding site for talin (94), paxillin (95) and p190RhoGEF (96). FAK-mediated tyrosine phosphorylation of Y12 in the α-actinin binding domain
negatively regulates the interaction between α-actinin and actin (97). FAK-deficient cells
show delayed spreading, reduced adhesion turnover (98), as well as decreased Rac activity
and loss of lamellipodia (99, 100). Over-expression of FAK on the other hand, increases the
directional motility of cells (101).
Another member of the of non-receptor tyrosine kinase family is the proline-rich tyrosine
kinase 2 (Pyk2; also designated CAKβ, RAFTK, FAK2, or CADTK) identified in 1995
23
(88-91). Pyk2 and FAK share high overall amino acid identity (48%) (102), and display
60% identity in the catalytic domain, 31% in the N-terminal part, and 45% at the Cterminus (102). Pyk2 is expressed mainly in the central nervous system and in cells of
hematopoietic origin (35, 103, 104). Two spliced isoforms of Pyk2 have been described,
characterized by the presence or absence of an exon coding for a 42 amino acid insert in
the C-terminal region (105, 106). Pyk2-H, the spliced variant lacking the exon, is
expressed in hematopoietic cells, and the unspliced form is predominant in the brain
(105). FAK has been detected in cell lysates of human neutrophils although it does not
appear to be activated by cell adhesion in these cells (107, 108). Differentiated HL60
cells do not express FAK (35). These data suggest that FAK has a less certain role in
integrin signalling in human neutrophils/HL60 cells (109), and we therefore propose
Pyk2 to be the focal adhesion protein tyrosine kinases involved in integrin mediatedsignal transduction in these cells.
The central catalytic domain of Pyk2 is flanked by non-catalytic regions on both sides.
The major autophosphorylation site is Tyr-402 in the N-terminal domain, which acts as a
binding site for the SH2 domain of Src (110). The activation of Pyk2 occurs in multiple
steps. The initial autophosphorylation of Tyr-402 (88, 111) mediates binding of Srckinases which in turn phosphorylate Tyr-580, thereby enhancing the enzymatic activity
of Pyk2 (112, 113). The proceeding phosphorylation of Pyk2 involves both Srcdependent and independent signalling pathways (110, 111, 114). The C-terminal domain
of Pyk2 contains two proline-rich motifs that function as sites for SH3-mediated proteinprotein interactions (115-120). Several proteins associate with Pyk2, including the focal
adhesion proteins; paxillin (121), and p130Cas (122), Src-kinases (102), the ARF-GAP
protein PAP (123), and the Nirs (124). Thus, Pyk2 not only functions as a link between
heterotrimeric G-protein-coupled receptors and signalling pathways (125, 126), but also
has an essential role in the transfer of signals from the adhesion receptors to regulators of
the cytoskeleton, i.e. serves as a scaffold in the formation/activation of focal complexes.
24
Cell motility
The capacity of cells to sense changes in the external environment during migration towards a
gradient of chemotactic substances (i.e. chemotaxis), is essential in inflammation. Neutrophils
are one of the fastest moving cells; migrating up to 10- 20 μm per minute, and represent a
good model system for investigating chemotaxis. Chemotaxis is initiated by binding of
chemoattractants to G protein-coupled receptors (GPCRs). GPCRs belong to a large family of
seven transmembrane spanning receptor proteins that activate intracellular heterotrimeric G
proteins consisting of α-, β-, and γ-subunit. Upon activation, the GDP bound to the α-subunit
is exchanged for GTP. GTP induces to dissociation of the β/γ and α subunit, and regulates
down-stream effectors, thereby amplifying the signal (Fig. 6).
The
chemoattractants
inducing
chemotaxis
are
N-formyl-L-methionyl-L-leucyl-L-
phenylalanine (fMLP) released by bacteria, the arachidonic acid metabolite leukotriene B4
(LTB4), complement factor 5a (C5a), platelet activating factor (PAF), and the chemokine
interleukin-8 (IL-8) (127). A hierarchy exists between the different chemoattractants in their
potency of inducing chemotaxis, where end-target chemoattractants e.g. fMLP are more
potent compared to intermediary chemoattractants e.g. IL-8. In a milieu of different
chemotactic gradients, the cells will favour end-target before intermediary chemoattractants
(128). Moreover, different signalling pathways are activated by intermediary, versus endtarget chemoattractants during neutrophil chemotaxis. The chemotactic response to end-target
chemoattractants involves p38 MAPK and CD11b/CD18, whereas the signalling pathways
activated by intermediary chemoattractants are dependent on PI-3K and CD11a/CD18 (128,
129).
25
GPCR
α γ
GTP β
?
PLC
Rho GTPases
(Rho, Rac, Cdc42)
PI 3K
MAPK
Tyrosine kinases
(Pyk2, Syk, Src)
PIP2
IP3
DAG
PIP2
PIP3
Calcium ↑ PKC
Figure 7. The signalling pathways activated by heterotrimeric G-proteins.
Chemoattractants, binding to seven transmembrane spanning, G protein-coupled
receptors (GPCRs), activate heterotrimeric G proteins. Upon GTP binding, the α- and
β/γ-subunits dissociate and mediate the activation of downstream effector proteins.
Adapted from (130).
Regulation of adhesion signalling by lipid metabolites
Phosholipase A2
To date, 22 genes encoding different phospholipase A2 (PLA2) proteins have been identified
in mammals (131). The superfamily of PLA2 can be divided, according to their biochemical
properties into 4 subfamilies: i) the Ca2+ -dependent secreted enzymes (sPLA2), ii) the Ca2+dependent cytosolic enzymes (cPLA2), iii) the Ca2+-independet cytosolic enzymes (iPLA2),
iv) and the platelet-activating factor acetyl hydrolases (PAF-AH) (132). It is generally
accepted that the activity of PLA2 is regulated by phosphorylation. Several kinases regulate
the activity of PLA2, including PKC and MAPK (133).
The plasma level of sPLA2 is increased in patients with inflammatory or autoimmune diseases
such as rheumatoid arthritis, septic shock, Crohn’s disease, and ulcerative colitis. In
26
neutrophils, the group IIA sPLA2 stimulates release of LTB4 (134), and increases the
expression of CD11b/CD18 by promoting translocation from secretory vesicles to the plasma
membrane (135). The unique trait for iPLA2 is the lack of requirement for calcium for their
enzymatic activity. iPLA2 is involved in regulation of cell physiology and metabolic
homeostasis, but is not a major player in inflammatory processes (136). Instead cPLA2 and
sPLA2 appear to be important for induction of inflammation.
Arachidonic acid and pro-inflammatory eicosanoids
In addition to regulation by protein-protein interactions, the signalling pathways mediated in
adherent and migrating leukocytes are heavily influenced by lipid mediators. Lipid mediators
have been found to enhance the β2-integrin mediated adhesion to fibrinogen and C3bi (137).
Moreover, β2-mediated phagocytosis induces the release of arachidonic acid (AA) (138).
Arachidonic acid is a polyunsaturated fatty acid hydrolyzed by PLA2 from the sn-2 position of
glycerophospholipids (139). Upon release, arachidonic acid per se can influence cellular
processes, e.g. actin bundling (140), degranulation, and superoxide production (141), or
become metabolized by either cyclooxygenases (COX) to prostaglandins and thromboxanes,
or by lipoxygenases (LOX) to form leukotrienes and lipoxins. The oxygenation of arachidonic
acid by 5-LOX, results in the formation of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), a
precursor for leukotrienes and lipoxins (Fig. 8). Various eicosanoids (AA metabolites, e.g. 5LOX metabolites), can regulate cell adhesion, as well as other processes involving
cytoskeletal reorganization (142, 143).
Activation of PLA2 and AA per se, increase the expression of β2-integrins on the plasma
membrane of neutrophils (144). cPLA2 and AA have also been shown to regulate the
superoxide production in phagocytes (145, 146). In addition, the arachidonic acid binding
protein S100A8/A9, has been identified as a novel binding partner for the NADPH-oxidase
subunits p67phox and Rac (146).
27
PLA2
Lyso-PAF
COOH
AA
PAF
5-LOX
5-HETE
COX-2
5-HPETE
PGH2
12-/15-LOX
LX
LTs
PGs
TXs
Figure 8. The arachidonic acid metabolism. Arachidonic acid (AA) is hydrolyzed
by phospholipase A2 (PLA2) at the sn-2 position of glycerophospholipids. The
lysophospholipid formed can be metabolized into platelet activating factor (PAF). AA is
metabolized via the cyclooxygenase (COX) pathway to prostaglandins (PGs) and
thromboxanes (TXs), or via lipoxygenase (LOX) pathways to leukotrienes (LTs), and
lipoxins (LX).
Anti-inflammatory lipoxins
Lipoxins are eicosanoids derived during a transcellular process by the combined action of 5LOX, and 12-LOX or 15-LOX (147). Lipoxins function as innate “stop signals” that control
inflammation processes (148) mainly by inhibiting the chemotactically induced signalling
pathways. Lipoxins inhibit the LTB4 and fMLP-induced chemotaxis (149), adhesion and
transmigration (150), expression of β2 integrins on neutrophils (151), and cytokine formation
(152). Lipoxins bind to G-protein-coupled seven-transmembrane spanning receptors. The
lipoxin receptor (ALX) belongs, together with fMLP- and LTB4-receptors, to the same cluster
of GPCRs (153).
In the presence of aspirin 15-epi lipoxins are formed, which are more potent than the native
lipoxins (153). Due to the rapid conversion of lipoxins to inactive compounds (154), stable
28
lipoxin analogues (LXa) have been synthesized (155). These analogues are almost as potent
as the aspirin-triggered lipoxins (ATL) (reviewed in (156)), and are therefore useful tools in
elucidating the signalling pathways induced by adhesion and chemotactic stimuli. The lipoxin
analogues inhibit PMN transmigration across epithelia and block PMN adhesion (155).
Lipoxins redistribute myosin IIA and Cdc42 in macrophages, and are implicated to regulate
phagocytosis and apoptosis (157) in a non-phlogistic manner.
Phagocytosis
Phagocytosis is defined as cellular engulfment of particles larger than 0.5 μm. Professional
phagocytes are cells mainly focusing on phagocytosis i.e. neutrophils, eosinophils, basophils,
monocytes, and macrophages. In order for phagocytosis to take place, the cells must first
localize, recognize and bind the particle. Recognition is mediated via specific receptors on the
plasma membrane. Two sets of receptor types are activated during phagocytosis, namely Fcreceptors (FcR) and complement receptors (CR) both mediating distinct signalling pathways.
Particles opsonized with IgG-antibodies are captured and engulfed by FcγR located on Factin-containing protrusions called pseudopodia. FcγR-mediated phagocytosis is dependent on
Ca2+ and actin reorganization. The FcγR-mediated signalling cascade involves the activation
of various proteins including: PLC, PKC, PI-3K, Rac, Cdc42, PLA2 and MAPK (reviewed
in(158)). The CR-mediated phagocytosis on the other hand, does not involve active formation
of pseudopodia and is thus less dependent on calcium. The complement receptor 3 (CR3),
identical to CD11b/CD18 integrin, binds C3bi-opsonized particles. An early event in CRmediated phagocytosis is tyrosine phosphorylation and activation of proteins such as: paxillin,
RhoGTPases, Pyk2, PLC, and vav (103, 107, 121, 159, 160).
The phagocytic process
Besides adhesion, migration, and aggregation, the phagocytic process also includes
engulfment of the particle, degranulation and bacterial killing. Killing of microorganisms
located in the phagosome, involves generation of reactive oxygen species (ROS) including
superoxide anion produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Superoxide anion produced by NADPH-oxidase, is converted by superoxide
29
dismutase (SOD) and catalase to H2O2 and H2O. In the presence of myeloperoxidase and
chloride, H2O2 forms the toxic hypocloric acid.
NADPH-oxidase consists of several membrane associated and cytosolic components, one of
them being the RhoGTPase Rac (161). Cdc42 has emerged as a competitor of Rac2 for the
binding to NADPH-oxidase (162, 163). The actin cytoskeleton plays an important role in
regulating the assembly and stabilization of the NADPH-oxidase (164, 165). Assembly of
NADPH-oxidase occurring at the membrane of intracellular components induces intracellular
ROS production, whereas activation of NADPH-oxidase at plasma membrane leads to
extracellular release of ROS.
Degranulation
Neutrophils are equipped with vesicles containing proteolytic enzymes and bactericidal
peptides to combat ingested bacteria. Four populations of granules are found in neutrophils
namely i) primary (azurophil) granules, ii) secondary (specific), peroxidase negative granules,
iii) tertiary gelatinase granules, and iv) secretory vesicles (reviewed in (166)). The different
granules are mobilized following a hierarchy, and differ in aspects of content and function.
The azurophilic granules contain for example myeloperoxidase, defensins, lysozymes, and
CD63. Certain receptors are also stored in granules and are upregulated upon stimulation. The
β2 integrins and receptors for fMLP and IL-8, localize to specific granules and/or secretory
vesicles (166). HL60 cells lack specific granules and the upregulation of for example β2
integrins is therefore impaired (167). Translocation of granules, and fusion with phagosome is
mediated by both the actin- and microtubulin network (168). The internalization of the
particle induces polymerization of F-actin at the site of ingestion. The F-actin surrounding the
phagosome must be degraded to allow endosome/lysosome to fuse with the phagosomal
membrane. Proteins implicated in the phagosomal maturation are SNAREs, rab 5, rab7, and
PKC (166, 168). Despite being activated during phagocytosis, neither Rac nor Cc42 has been
found to localize to the plasma membrane in their active form. Since small GTPases regulate
actin turnover, the involvement of activate Rac and Cdc42 in phago-lysosomal formation
could be expected.
30
AIMS OF THE INVESTIGATION
The aim of this thesis was to shed light on the regulation of the β2 integrin-mediated
signalling in human neutrophils. To achieve this, pro- inflammatory (i.e. fMLP, IL-8 and
LTB4) substances, and anti-inflammatory endogenous- (i.e. lipoxin, epi-lipoxin), and
exogenous substances (i.e. statin, aspirin) were employed. The more specific aims were to
understand:
i)
The regulatory effect of pro- and anti-inflammatory substances on the β2 integrinactivation state (avidity vs. affinity) of human neutrophils from healthy donors and
from patients suffering from coronary artery disease (CAD).
Statin
β2 integrins
LTB4-R
fMLP-R
IL-8-R
?
LXA4-R
ii)
Aspirin
The regulatory effect of the pro- and anti-inflammatory substances, on the signalling
capacity of β2 integrins (i.e. the release of AA, and production of ROS), in healthy
donors and in patients suffering from coronary artery disease (CAD).
31
+ aspirin, lipoxin, statin ?
β2 pansorbin
fMLP
LTB4
?
?
AA
ROS
iii)
The effect of the pro- and anti- inflammatory substances on the β2 integrin-mediated
signal transduction (phosphorylation of Pyk2) in neutrophils.
+ aspirin, lipoxin ?
β2 pansorbin
fMLP
LTB4
?
?
Pyk2
iv)
P
In addition, the aim was to establish methods for investigating the proteins involved
in
the
regulation
of
β2
integrin-mediated
signalling,
e.g.
western
blot,
immunoprecipitation, immunofluorescence, single particle tracking, and TATtransduction.
32
METHODS
Some of the methods used in this thesis will be discussed briefly herein.
Neutrophils
When studying blood cells, different approaches are available. One is to use freshly isolated
neutrophils from blood donors. The limited usage of neutrophils lies in the availability of the
cells and in the limited life span of the neutrophils once isolated from whole blood. The status
of the blood cells from donors can also vary between experiments, due to individual donor
differences. Moreover, the neutrophils are affected by the isolation procedure, and the use of
whole blood is therefore preferable for some assays. However, when interpreting the results,
one must keep in mind that the different cell types in whole blood could influence each other.
In his study, whole blood was used when analyzing neutrophils from patients suffering from
stable Coronary Artery Disease (CAD), due to the limitation in blood sample volumes.
Thirty patients with angiographically verified stable CAD were recruited. Each patient was
matched, regarding age and gender, with a clinically healthy control subject, randomly
selected from the population register. Patients were excluded if they were >65 years old, had
severe heart failure, immunologic disorders, neoplasm disease, evidence of acute or recent (<2
months) infection, recent major trauma, surgery or revascularization procedure, or treatment
with immunosuppressive or anti-inflammatory agents (except low-dose aspirin).
Another approach when studying neutrophils is to use commercially available cell lines,
which can be differentiated into neutrophil-like cells. The cells have a more stable phenotype
compared to freshly isolated neutrophils. However, the tumour cells may have some inherent
defects. In this thesis, we have used both freshly isolated human neutrophils, and a
promyelocytic cell line i.e. Human Leukemia 60 (HL60). The HL60 cells can be
differentiated into neutrophils, monocytes and macrophages depending on the differentiation
agent used. When grown in suspension in the presence of DMSO for 6-7 days (169), the
HL60 cells become neutrophil-like and acquire the ability to adhere, migrate, and
33
phagocytose like the peripheral blood neutrophils. Neutrophil-like HL60 cells do not contain
specific granules and lack the ability to up-regulate some receptors on the plasma membrane.
This can be used as tool when studying certain cellular processes in neutrophils, such as the
activation state (affinity /avidity) of β2 integrins.
[3H]arachidonic acid-labelling and release
To investigate the role of lipid mediators in β2 integrin signalling, the cells were incubated
with [3H]labelled arachidonic acid ([3H]AA). Isolated neutrophils were incubated for 2h at
37°C, whereas HL60 cells were labelled for the final 18-24 hours of the cell-differentiation
period. During the experiments, human serum albumin (HSA) was added in order to trap the
released fatty acids and prevent further metabolism (170). The reaction was stopped by
centrifugation and the radioactivity of the supernatant was measured in a β-counter.
Cell stimulation
Activation by chemotactic factors
During inflammation, neutrophils navigate along a gradient of chemotactic factors e.g. fMLP,
LTB4, and IL-8. fMLP is an end-target chemoattractant released by the bacteria at the site of
infection, and is considered more potent than the endogenously formed intermediary
chemoattractants LTB4 and IL-8 (128). In this study, the effect of intermediary- and end target
chemoattractants on β2 integrin signalling was investigated both in differentiated HL60 cells
and in human neutrophils from healthy donors and CAD patients.
Integrin ligation
Activation by antibody-coated Pansorbins®
The pansorbins® are heat-hardened Staphylococcus aureus, expressing protein A on the
surface. The Fc-part of the antibody will bind to protein A, giving rise to a pansorbin particle
coated with antibodies exposing the Fab-part outwards, in contrast to opsonized particles (Fig.
9). By using pansorbins, receptors on the plasma membrane of cells kept in suspension can be
directly activated without the interference from other adhesion receptors. This is a good
model system for β2 integrin ligation, since the particles do not mediate spreading or
34
phagocytosis. One advantage of using Pansorbins® is that the unspecific antibody-interactions
are reduced, since the Fc-part of the antibody is coupled to the Pansorbin® and can not
interact with Fc-receptors.
A
B
Intracelullar signalling
Phagocytosis
and signalling
Figure 9. A) The difference between antibody-coated and antibody-opsonized particles.
Anti-β2 integrin antibodies coupled to pansorbins® interact with β2 integrin receptors via
their Fc-part, giving rise to receptor mediated signalling. B) Pansorbins® opsonized with
IgG antibodies are recognized by the receptors through the Fc-part of the antibody,
inducing Fc receptor-mediated phagocytosis and subsequent signalling.
Integrin ligation in adherent cells
In order to verify the results observed by anti-β2 integrin-pansorbins, cells were allowed to
adhere to the β2-integrin ligand, fibrinogen. Using the adhesion assay enabled us to study the
signalling pathways induced by β2 integrin ligation and reorganization of the cytoskeleton,
and to elucidate the need for “cellular priming”, i.e. integrin activation, prior to adhesion. As a
control, cells were plated on BSA-coated culture dishes.
Flow cytometry
Flow cytometry is a quantitative approach, used to analyze the total fluorescence of individual
cells/particles ranging from 0.5 μm to 40 μm in size, in a single cell suspension. Intracellular
molecules and membrane markers can be stained with different fluorochromes and analyzed
simultaneously in the flow cytometer (Fig. 10). The cells pass through a light source one at a
time. Light is scattered upon passing through the cells and the fluorochromes are exited to a
35
higher energy state. This energy released in form of emitted light, is detected together with the
scattered light by several detectors. Light passing through the cells is detected as forward
scatter (FSC) and is relative to the cell size. The more granular the cells, the more the light is
scattered to the sides, detected as side scatter (SSC). By plotting FCS against SSC, different
cell populations can be visualised and a region of interest established, so called gating.
Therefore, when analyzing different blood cells by flow cytometry, whole blood can be
analyzed directly in the flow cytometer. The cell population of interest is then visualized in a
frequency histogram and the mean fluorescence intensity (MFI) can be analyzed by statistics.
B
SSC
A
Dot Plot
Region of interrest
(gating)
Detectors
Laser
FSC
Forward scatter
(FSC = cellular size)
Fluorescense
Number of events
(FL1)
Side scatter
(SSC = granularity)
Frequency histogram for the population
MFI
FL1
Movement of the curve to the right is
equivalent to an increase in MFI
Figure 10. The principles of flow cytometry. In brief; cells suspended in a carrier
solution, pass a light source one at a time, and the fluorochromes become excitated to a
higher energy state. The light emission is detected together with the scattered light,
enabling the selection of a region of interest (i.e. gating). Fluorescence (FL1) is plotted in
a frequency histogram and the mean fluorescence intensity (MFI) is calculated for the
selected population.
In this study, the FITC-fluorescence of individual cells was determined by flow cytometry,
using a Becton Dickinson FACSCalibur (Becton Dickinson, San Jose, CA/USA). Mean
36
fluorescence values, from a minimum of 5000 cells/ sample, were determined. Gating was
performed by using forward scatter versus side scatter (excluding cell debris).
Autofluorescence of unstained cells was routinely analyzed and subtracted from the
fluorescence values of the stained cells. Unspecific binding was analyzed by isotype
antibodies.
Expression of β2-integrins
Prior to investigating the activation state of the β2 integrins, it was important to quantify the
number of integrin receptors on the plasma membranes of HL60 cells. If no receptors are
upregulated upon stimulation, increased integrin affinity should be due to an activation of β2
integrins and not be dependent of an increased number of receptors on the plasma membrane.
In brief: Cells were pre-incubated with anti and pro-inflammatory stimuli alone or in
combination. Stimulation was stopped by ice-cold PFA, washed and incubated with primary
anti-CD18 antibodies, followed by incubation with secondary, FITC-conjugated antibodies.
In human neutrophils, β2 integrins are stored in granules and become expressed on the
membrane upon stimulation. The basal expression of β2 integrins on human neutrophils from
healthy donors and CAD patients was therefore investigated. Due to limitations in blood
volume from patients, the flow cytometry assay was adjusted by analysing neutrophils from
whole blood.
β2 integrin affinity
One way of investigating the activation state of the integrins is to measure affinity of the
integrin receptors. Two high-affinity antibodies; CBRM 1/5 and MAb24 were used in this
study. CBRM 1/5 is a monoclonal antibody recognizing the ligand binding domain (i.e. Idomain) on CD11b integrins (171), whereas MAb24 recognizes a high affinity epitope on the
α-subunit of both the CD11a and CD11b integrins (172). In order to measure affinity of β2
integrins, the antibodies had to be present prior to stimulation of the cells, probably due to
destruction of the recognition epitopes by PFA. The cells were pre-incubated with the CBRM
1/5 antibody for one minute prior to stimulation, fixed in PFA and incubated with FITC-
37
conjugated secondary antibodies. Alternatively, a FITC-conjugated CBRM 1/5 antibody was
used.
Binding of soluble ICAM-1
The β2 integrin-ligand ICAM-1, binds to the receptor when the ligand binding domain is
exposed upon increased affinity. In order to verify the results obtained by the high affinity
antibody with a more biological read-out, we investigated the binding of soluble ICAM-1 (sICAM) to the β2 integrins (173) by flow cytometry.
Fluorescence microscopy
The fluorescence microscope was used to evaluate the lateral movement of β2 integrins
during the Single Particle Tracking experiments, and to analyze the distribution of F-actin in
HL60 cells during phagocytosis. In contrast to flow cytometry, which gives the total amount
of fluorescence in a cell, the fluorescence microscope can be used to determine the
distribution of fluorescence in the cell.
Single Particle Tracking
The Single Particle Tracking (SPT) technique was used to measure the movement of
individual β2 integrins on the plasma membrane of neutrophils. By conjugating β2 integrinspecific antibodies to fluorescent latex beads (174), the lateral movement of integrin receptors
can be monitored in a fluorescence microscope (Fig. 11). In short: neutrophils were plated on
poly-L-lysine-coated cover slips and stimulated. The movement of individual antibodyconjugated beads was recorded for 30 seconds and the diffusion coefficient (D) was
calculated.
38
Membrane receptor
Fluorescent
latex bead (conjugated
with a receptor specific antibody)
Lateral movement
of receptors
Analysis in a fluorescence microscope
= Diffusion Coefficient
(calculated by software)
Figure 11. The principles of the single particle tracking method. Cells are
stimulated and allowed to settle on poly-L-lysine coated cover slips. The cells are
incubated with specific antibodies conjugated to fluorescent latex beads, and movement of
the fluorescent particles is monitored in a fluorescence microscope. The diffusion
coefficient, equivalent to lateral movement of the receptor, is determined by computer
software.
Confocal Scanning Light Microscopy
By using Confocal Scanning Light microscopy (CSLM), the fluorescence from a thin optical
section in a cell (a focal plane) can be analyzed. In contrast to ordinary fluorescence
microscopy, CSLM gives a high resolution image of a chosen focal plane in a cell,
simultaneously eliminating the light coming from out-of-focus regions above and below that
plane. CSLM was used to analyze clustering of β2 integrin receptors on the plasma
membrane, the actin distribution around the phagosome, and the distribution of CD63-positive
granules in HL60 cells.
39
Image analysis
To analyze the results obtained by CSLM different imaging analysis procedures were used.
Clustering of integrins
In order to quantify the size of the integrin clusters from a confocal microscope image, the β2
integrins on individual cells were traced manually. The fluorescence was plotted as a
histogram in which the peaks were equivalent to clusters of integrins. This allowed us to
quantify the number of small and large peaks i.e. small/large β2 integrin clusters.
Granule distribution
The effect of TAT-proteins on the distribution of CD63 positive granules was analyzed by
CSLM. To more objectively evaluate granule movement in living cells, confocal microscope
images of cells loaded with LysoTracker®Red DND 99 were analyzed. Software was used to
evaluate the accumulation-, number-, and distribution of granules in the cells.
Introduction of recombinant proteins into human neutrophils
Transfection is a method used to introduce recombinant DNA into cells using eukaryotic
expression vectors. The most common transfection methods are electroporation, liposomebased transfection (e.g. Lipofectamine), retroviral gene transfer, cationic polymer-based
system (e.g. DEAE-dextran), and the use of chemicals such as CaPO4 (175, 176). DNA can
also be directly injected into a cell, by so called microinjection. When working with
neutrophils, one quickly discovers the obstacles lying ahead. Neutrophils have a limited life
span, and contain granules filled with degrading enzymes. The production of stable
transfectants is therefore impossible. To circumvent this, cell lines can be used for
transfection. Initially in this project, efforts were made to transfect neutrophils and HL60 cells
by various transfection approaches (i.e. electroporation, DEAE-dextran, retrovirus, and
lipofectamine) without success, hence other methods to introduce recombinant proteins were
established.
40
HIV Tat proteins
The human immunodefiency virus-1 (HIV-1) contains a transcriptional activator of
transcription (Tat) necessary for the replication of the virus (reviewed in (177)). In 1988, it
was shown that the HIV-tat protein (86 amino acids) could cross cellular membranes and
accumulate in the nucleus and induce gene activation (178, 179). Different molecules linked
to Tat, e.g. FITC, DNA and proteins, translocate along with the Tat-peptide into the
cytoplasm. The uptake of tat-proteins into the cell remains unclear, but is proposed to take
place via a receptor/transporter- and endocytosis- independent pathway. Tat-proteins have
also been suggested to directly penetrate the membranes (180), via a process facilitated by,
but not dependent of, denaturation and refolding of the proteins (181, 182).
Nevertheless, Tat-mediated protein transduction has emerged as a useful tool to insert
molecules into cells, in which transfection protocols are not applicable.
TAT transduction
In this study, we used a pTAT- HA vector (181) containing an amino-terminal, transduction
domain of HIV Tat (11 aa), termed TAT. The pTAT-HA vector contains the gene for six
histidines (used for affinity purification) and a hemagglutinine (HA) epitope (used for
detection of transduced proteins) linked to a TAT-sequence, followed by a multiple cloning
site (Fig. 12). Initially, human neutrophils were transduced with TAT-fusion proteins, but due
to rapid degradation of the proteins, differentiated HL60 cells were used instead. After
optimization of the transduction conditions, the cells were transduced with 200 nM TATfusion proteins for 30 min at 37ºC in calcium free-KRG. The transduction efficiency was 65%
or more.
YGRKKRRQRRR
Figure 12. Characterization of the
pTAT-expression vector.
The pTAT-HA vector contains six
histidines (His6), followed by 11 amino
acid transduction domain of HIV-Tat
(TAT), and a hemagglutinine (HA) epitope
linked to a multiple cloning site (MCS).
Adapted from (181).
ATG-His 6-TAT-HA-MCS
pTAT-HA
41
Immunoprecipitation and Western blot
When studying activation of intracellular proteins different approaches can be used. The first
step involves mechanical (e.g. sonication, homogenization), or chemical (e.g. Triton-X 100,
saponine) disturbance of the plasma membrane integrity in order to access the intracellular
proteins. The proteins of interest can be “fished out” by specific antibodies conjugated to
sepharose beads (i.e. immunoprecipitation), and subjected to SDS-PAGE and Western Blot.
During SDS-PAGE, proteins are electrically separated on a polyacrylamide gel according to
size and charge, transferred to nitrocellulose membrane and incubated with antibodies
(Western Blot; WB).
The advantages of using immunoprecipitation instead of whole cell lysate is that i) the
number of “bands” on the gel is limited giving a “purer” blot, ii) due to amplification of the
proteins larger quantities can be loaded on the gel, iii) associated proteins can be detected
after stripping and re-probing with additional antibodies.
In this study, we have used anti-Pyk2 antibodies together with phosphotyrosine antibodies, to
immunoprecipitate Pyk2 and screen for phosphorylated Pyk2 proteins.
Phagocytosis and production of reactive oxygen species
Different approaches can be used to quantify phagocytosis. The amount of fluorescent
conjugated particles, associated to the cells can be quantified by flow cytometry. Using light
microscopy, phagocytosis can be estimated directly. Phagocytosis, especially FcR-mediated,
has been shown to be a good model system for studying adhesion signalling (reviewed in
(183)), and was therefore used as a biological read-out to evaluate the involvement of Cdc42
in this study. The effect of GTPases on phagocytosis and phagocytosis-mediated signalling
was evaluated by light and fluorescence microscopy.
Statistical analysis
Student´s t-test was used to calculate the significance of results displaying a Gaussian
distribution. When data lacked Gaussian distribution, a non parametric test was chosen i.e.
Mann Whitney test when comparing two groups, or Kruskal-Wallis one-way analysis of
variance by ranks together with Dunn’s post test. All the statistics were calculated using
42
GraphPad InStat version 3.0.1 (www.graphpad.com).* represents p <0.05, ** represents
p<0.01, and *** p< 0.001.
43
RESULTS
The effect of pro- and anti-inflammatory chemoattractants on the
activation state of the β2 integrins
The activation state of β2 integrins on neutrophils from healthy donors,
mediated by pro-inflammatory chemoattractants
One aim of this thesis was to elucidate the effect of pro-inflammatory substances e.g. fMLP,
LTB4, and IL-8 on the activation state of β2 integrins in human neutrophils.
Upon activation, the connection between integrin receptors and the cytoskeleton is
momentarily broken (60), allowing lateral movement of the receptors in the plasma membrane
(184). β2 integrins cluster on the membrane and form focal complexes to which structural and
signalling molecules are recruited, thereby enhancing the avidity of the receptor for its ligand,
and the signalling capacity. Analysis of the mobility and clustering of integrin receptors on
the plasma membrane showed that the chemoattractant LTB4 induced an increase in β2
B
integrin mobility and the formation of both small and large clusters (Paper I, and Fig. 14). The
chemoattractant fMLP on the other hand, was less potent compared to LTB4 in mediating
lateral mobility of the integrins and induced formation of predominantly small β2 integrinclusters on neutrophil-like HL60 cells (Paper I, and summarized in Fig.14).
In order to determine the affinity status of β2 integrins, two α-integrin antibodies were used i)
an antibody recognizing a high affinity epitope on the α-subunit of both CD11a and CD11b
integrins (MAb24) (172), and ii) antibodies recognizing the ligand binding domain (I-domain)
on CD11b integrins (CBRM 1/5). It has been disputed whether the results obtained by these
antibodies correlate with ligand binding or not (185). Therefore, our results were confirmed
by measuring the binding of soluble ICAM (s-ICAM), a natural ligand to β2 integrins. The
binding studies confirmed the results on β2 integrin affinity showing that fMLP, but not
LTB4, induced an affinity change of the β2 integrins (Paper I).
Lately, arachidonic acid, the pre-cursor of both pro-and anti- inflammatory eicosanoids, has
emerged as a mediator of inflammatory responses. In HL60 cells, arachidonic acid induced a small
44
release of [3H]AA and mediated an increase in β2 integrin affinity (Paper III). Arachidonic acid
also induced an increase in the avidity of β2 integrins in differentiated HL60 cells (Fig. 13,
unpublished results), supporting a role of arachidonic acid as mediator of the activation of β2
integrins.
A
250
AA 20uM
AA 100uM
NKLI16 MFI (% of control)
225
200
175
150
125
100
75
50
0
5
10
Time (min)
Figure 13. The effect of arachidonic acid on the β2 integrin avidity in
differentiated HL60 cells. Differentiated HL60 cells were stimulated with 20 μM or
100 μM of arachidonic acid (AA) for various time-points. The cells were fixed in PFA for
15 minutes and incubated with NKLI16 antibodies recognizing clustered integrins.
Increase in integrin avidity was evaluated by flow cytometry. Data are expressed as % of
control from one experiment.
The effect of anti-inflammatory substances on the activation state of β2
integrins
The anti-inflammatory, exogenous lipoxin analogues (LXa) were used as a tool for
elucidating the signalling pathways that regulate the β2 integrin activation and to investigate
the effect of anti-inflammatory substances per se. LXa inhibited the formation of small fMLPmediated clusters by half, but had no effect on the LTB4-mediated formation of small clusters
(Paper I). The large, LTB4-mediated β2 integrin clusters were sensitive to LXa-treatment.
B
LXa had no effect on the fMLP-induced, β2 integrin affinity (Paper I and summarized in Fig.
14). In conclusion, LXa seems to regulate the activity of β2 integrins mainly by affecting the
avidity of the adhesion receptors.
45
Lateral mobility
Clustring
Affinity
+ ligand binding
ICAM-1
β2 integrins
Small
clusters
Large
clusters
no effect
fMLP
+
++
(+ LXa)
(-)
(- -)
+++
LTB4
+++
++
+
+
(+ LXa)
(- -)
(-)
(-)
(no effect)
(no effect)
Figure 14. The effect of fMLP and LTB4, on the activation state of β2
integrins on neutrophils. A schematic illustration of the results presented in Paper I.
The fMLP induced increase of integrin affinity and ligand binding is insensitive to LXa
treatment. LTB4 on the other hand, has only a weak effect on β2 integrin affinity, but a
strong effect on both the mobility of β2 integrins, as well as clustering. LXa inhibits the
LTB4-mediated mobility and clustering of β2 integrins but has no effect on the LTB4mediated β2 integrin affinity. + represents weak positive effect; ++ intermediary effect;
and +++ strong positive effect. The effect of LXa on the fMLP- and LTB4-mediated
activation of β2 integrins is given in parenthesis. – represents weak inhibition, and – –
represents a strong inhibition.
B
The activation state of β2 integrins on neutrophils from patients with
stable CAD
After analyzing β2 integrin mediated signalling in under normal conditions, the signalling
capacity of neutrophils from patients suffering from stable coronary artery disease (CAD) was
investigated. Circulation of leukocytes and extravasation to the tissues is one part of the
normal inflammatory response. Abnormal accumulation of leukocytes in target tissues is
connected to a variety of diseases such as: rheumatoid arthritis, multiple sclerosis and
atherosclerosis (186). The activation state of β2 integrins was investigated by quantifying the
number of β2 integrins on the plasma membrane as well as the affinity of the β2 integrin
receptors on neutrophils from CAD patients compared to age and gender matched healthy
donor subjects. The medication administered to all CAD patients consists of a low dose
46
aspirin combined with statins. Statins affect a number of signalling molecules important for
adhesion (187), and aspirin induces the formation of endogenous, anti-inflammatory
substances, i.e. 15-epi lipoxins, earlier found to inhibit the avidity state of the β2 integrins
(Paper I). Our study showed that there was no difference between patients and control
subjects, in neither the expression-, or in the affinity of β2 integrins during basal conditions
(Paper V). The lack of a primed status of neutrophils shows that the therapy of today is
sufficient in maintaining the neutrophils in a non-reactive state, thereby limiting progress of
the disease.
The effect of pro- and anti- inflammatory substances on the
signalling capacity of β2 integrins
Signalling capacity, as measured by the release of arachidonic acid
After investigating the activation state of β2 integrins on neutrophils from healthy donors and
CAD patients, the aim was to look more closely at the signalling capacity of the β2 integrin
receptors.
Arachidonic acid (AA) is a poly-unsaturated lipid incorporated in cell membranes, which is
released upon stimulation and metabolized into eicosanoids. In other words, arachidonic acid
is a precursor for both pro-inflammatory (e.g. LTB4) and anti-inflammatory (lipoxins)
eicosanoids. Starting this investigation, AA had been found to be released in HeLa cells
adherent to collagen (188). In macrophages on the other hand, CR3-mediated (β2 integrinmediated) phagocytosis did not activate arachidonate release (189). We found that in HL60
cells kept in suspension, ligation of β2 integrins by pansorbins induced a release of [3H]AA
(Paper III, and summarized in Fig. 15A). fMLP by itself, induced a substantial release of
[3H]AA, and enhanced the release mediated by β2 integrin-ligation probably due to inside-out
signalling (Paper III, and summarized in Fig. 15A). Depletion of calcium affected the AA
release, probably due to the fact that calcium is necessary for the activation of PLA2 and for
the activation of β2 integrins per se (Paper III). The intermediary chemoattractant LTB4 on
the other hand, did not induce any release of [3H]AA in differentiated HL60 cells (Fig. 15B,
unpublished results, and summarized 15A).
47
A
450
β2 pansorbin
B
[3H]AA release (% of control)
400
fMLP
LTB4
+
AA
350
300
250
200
150
100
50
Increase of: β2 integrin affinity and avidity,
amount of F-actin, and fillipodia formation
0
fMLP
LTB4
Figure 15. The effect of fMLP and LTB4 on the release of arachidonic acid in
differentiated HL60 cells. A) A schematic illustration on the effect of fMLP and
LTB4 on the β2 integrin-mediated release of arachidonic acid in HL60 cells. In contrast
to LTB4, both fMLP and β2 integrin-pansorbins induce the release of [3H]AA. Preincubation of fMLP potentiates the fMLP-mediated release of AA. Data are presented in
Paper III. B) The release of [3H]AA in differentiated HL60 cells. HL60 cells were
incubated with [3H]AA for 18-24 hours. The cells were washed and pre-incubated for 1
minute prior to stimulation with fMLP (100 nM) or LTB4 (100 nM) for 5 minutes. The
stimulation was stopped by centrifugation, and the radioactivity of supernatant was
measured in a β-scintillation counter. For more details see Materials and methods section
in Paper III. Data shown are expressed as mean ± SEM from 4 separate experiments, run
in duplicates.
Arachidonic acid, released via active β2 integrins and by fMLP, could become further
metabolized into eicosanoids, or affect biological processes per se. Plasma levels of free
arachidonic acid vary between 6-50 μM (190), but can under pathological conditions increase
to both 100 μM (malaria) and 500 μM (brain ischemia) (191). A low concentration of
arachidonic acid (20 μM) had no effect on cellular morphology and F-actin-content in HL60
cells (Fig. 16 and 17, unpublished results). Cells exposed to 100 μM of arachidonic acid on
the other hand, displayed a more round morphology with extensive fillipodia formation (Fig.
16, unpublished results), and increased level of F-actin (Fig. 17, unpublished results).
48
A
B
AA
20μM
AA
100μM
Figure 16. The effect of arachidonic acid on the cellular morphology of HL60
cells. Differentiated HL60 cells were pre-incubated with arachidonic acid (AA) prior to
plating for 15 minutes on glass slides pre-coated with IgG-opsonized yeast. Incubation
was stopped by addition of ice-cold PFA, followed by permeabilization and staining of Factin in PBS supplemented with lysophosphatidylcholine and Alexa 594-conjugated
phalloidin. Panels A) show HL60 cells during spreading and chemotaxis, and B) during
phagocytosis. The data shown are representative of two independent experiments.
B
F-actin MFI (% of control)
300
250
200
150
100
AA
fMLP
Figure 17. The effect of arachidonic acid on the amount of F-actin in
differentiated HL60 cells. Differentiated HL60 cells were stimulated with 100 μM
arachidonic acid (AA) or 100 nM fMLP and fixed in PFA for 15 minutes. F-actin was
labelled with FITC-conjugated phalloidin and evaluated by flow cytometry. Data are
expressed as median values from two independent experiments.
49
The signalling capacity of β2 integrins, measured by production of
reactive oxygen species
The signalling capacity of β2 integrins was determined by analyzing the effect of the proinflammatory chemoattractants LTB4 and IL-8, on the C3bi- (i.e. β2 integrin) mediated ROSproduction in neutrophils from healthy donor subjects. Compared to the β2 integrin-mediated
intracellular release, the chemoattractants mainly induced a extracellular production of ROS
(Paper V, and summarized in Fig. 18A).
It has been proposed that the progress of disease is due to a primed state of neutrophils.
However, the signalling capacity of neutrophils from CAD patient was decreased compared to
healthy donors, i.e. the neutrophils from CAD patients were less prone to produce ROS when
challenged with C3bi-opsonized yeast or PMA (Paper V, and summarized in Fig. 18B). We
hypothesize that the impaired response to C3bi-opsonized yeast in CAD-patients could result
from decreased adhesion brought about by a decreased β2 integrin avidity due to the antiinflammatory medication administered to CAD patients.
50
Aspirin
LTB4
O2-
LTB4
IL-8
O2-
O2
O2 O2-
Statin
IL-8
O2-
O2
O2
O2-
A
O2 O2-
O2
B
Figure 18. A schematic illustration over the production of reactive oxygen
species in human neutrophils. A) The chemoattractants; LTB4 and IL-8, mainly
induce extracellular production of reactive oxygen species (ROS) in neutrophils from
healthy donor subjects. The production mediated by β2 integrins on the other hand, is predominantly intracellular. B) The β2 integrin-mediated production of ROS is impaired in
human neutrophils from CAD patients, which are given statins and aspirin. For more
details see paper V.
The effect of pro- and anti-inflammatory substances on the signal
transduction mediated by β2 integrins
After investigating the mobility, clustering, and affinity of β2 integrins, the focus was turned
to their signal transduction. This process involves β2 integrin- mediated scaffolding of
cytoskeletal proteins and signalling molecules, and utilizes the cytoskeleton as a matrix for
the interactions. One of the signalling events activated by ligation of β2 integrins is
phosphorylation of proteins mediated by protein tyrosine kinases. When this investigation was
initiated in 1998, FAK had been detected in cell lysates of human neutrophils, but was not
activated by adhesion in these cells. Another member of the family of non-receptor protein
tyrosine kinases, Pyk2, was expressed in hematopoietic cells (104). The aim was therefore to
investigate the role of Pyk2 in β2 integrin-mediated signalling in HL60 cells. The strong
51
tyrosine phosphorylation of Pyk2 induced by fMLP and ligation of β2 integrins in cells kept
in suspension was mediated by Src and PKC (Paper II, and summarized in Fig 19) (35, 103).
The chemoattractant LTB4 on the other hand, did not induce phosphorylation of Pyk2 at any
B
given time or concentration. Adhesion to fibrinogen also induced phosphorylation of Pyk2 in
HL60 cells, although to a lesser extent compared to β2 pansorbins (Paper II). This can be
explained by the ability of the pansorbins to cross-link integrin receptors in the plasma
membrane, thereby mimicking clustering/scaffolding of the integrins. Moreover, preincubation with fMLP or LTB4 prior to adhesion, increased the integrin-mediated Pyk2
phosphorylation, most probably due to pre-activation of β2 integrin receptors by inside-out
signalling (Paper II, and summarized in Fig. 19). LXa inhibited fMLP-induced
phosphorylation of Pyk2 in cells kept in suspension, but did not affect the β2 integrin
mediated Pyk2 phosphorylation in cells adherent to fibrinogen (Paper II). Our results
concerning the effect of fMLP and LTB4 on the lateral mobility, clustering, affinity and
signalling capacity of β2 integrins (summarized in Fig. 14), support the idea that different
chemoattractants activate distinct signalling pathways during β2 integrin mediated signalling
(128, 129). Lipoxins emerge as potential anti-inflammatory drugs, and Pyk2 may be a suitable
target for the action of these drugs.
52
Inhibition
Activation
β2 pansorbin
fMLP
LTB4
?
LXa
Pyk2 P
Figure 19. Schematic illustration about the effect of chemotactic stimuli on
β2 integrin-mediated adhesion in neutrophils. The effect of fMLP and LTB4 on
the phosphorylation of Pyk2 in HL60 cells in suspension. Ligation of β2 integrins by
pansorbins induces a phosphorylation of Pyk2 in differentiated HL60 cells. The
chemoattractant fMLP phosphorylates Pyk2. Due to the strong activation of Pyk2 by β2pansorbins, it is impossible to evaluate the effect of fMLP on the pansorbin-mediated
phosphorylation of Pyk2. Pre-incubation with the anti-inflammatory lipoxin analogues
(LXa) inhibits fMLP-mediated activation of Pyk2. Data are presented in detail in paper
III.
53
Since arachidonic acid was found to mediate the activation status of β2 integrins (Paper III),
the effect of arachidonic acid on the phosphorylation of Pyk2 was investigated. Stimulation
with arachidonic acid (100 μM) had no effect on the phosphorylation of Pyk2 (Fig. 20,
unpublished results).
IP:Pyk2
-
+
-
- AA
+ fMLP
WB:4G10
WB:Pyk2
Figure 20. The effect of arachidonic acid on the phosphorylation of Pyk2 in
differentiated HL60 cells. Serum starved HL60 cells were stimulated with arachidonic
acid (AA; 100 μM) or fMLP (100 nM) for 2.5 minutes and lysed in ice-cold lysis buffer.
The cells were centrifuged at 12000 x g for 10 minutes, and Pyk2 in the supernatant was
immunoprecipitated (IP) by anti-Pyk2 antibodies (#600) together with protein A
sepharose. The samples were subjected to SDS-PAGE, and western blot (WB). After
blocking, the membranes were incubated with phosphotyrosine antibodies (4G10). In
order to ensure proper loading, membranes were stripped and incubated with anti-Pyk2
antibodies.
These results are supported by our finding that the AA-metabolite, LTB4 does to induce
B
phosphorylation of Pyk2 (Paper II), in likeness of its precursor AA. Additional experiments
are however needed to understand the effect of arachidonic acid on the activation of β2
integrin mediated signalling.
Additional methods to study proteins important for the regulation of
β2 integrins
In order to claim a protein to be of importance for a cellular process, the effect of inhibition of
the protein must be investigated. Pyk2 was earlier proposed to be a protein involved in the
signalling mediated by β2 integrins. No specific inhibitors of Pyk2 were commercially
available when this study was initiated, but the effect of tyrosine kinase inhibitors suggested
involvement of Pyk2 in β2 integrin-mediated signalling (Paper II). A way to introduce
constitutively active and dominant negative, recombinant forms of Pyk2 was therefore
54
needed. Transfection of neutrophils/HL60 cells by; Lipofectamine, DEAE-dextran,
electroporation, and retro-viral approach was however unsuccessful, hence other transfection
methods needed to be established.
TAT-protein transduction
The TAT-transduction method which utilizes short sequence of HIV-Tat protein expressed as
a fusion protein was exploited. Due to the small size of Cdc42 (approx. 25kDa), it was
considered as a suitable protein for optimizing the transduction method in neutrophils and
differentiated HL60 cells (Paper IV). Moreover, the fact that Cdc42 is activated by both β2integrin ligation and stimulation with both fMLP and LTB4 in human neutrophils (data not
shown), and that G-proteins are involved in the activation of Pyk2, made Cdc42 highly
interesting to investigate.
Enzymatic degradation of TAT-Cdc42 fusion proteins in human neutrophils forced us to
focus on differentiated HL60 cells. After optimizing the procedure, differentiated HL60 were
successfully transduced with TAT-Cdc42 (Paper IV).
As a biological read-out, the effect of constitutively active- (V12) and dominant negative(N17) forms of TAT-Cdc42 on IgG-mediated phagocytosis was analyzed. Staining of F-actin
by phalloidin, revealed that cells transduced with constitutively active TAT-Cdc42 displayed
a pronounced accumulation of F-actin, both at the leading edge of the cell and around the
phagosome (Paper IV, and in Fig. 23), which was also confirmed by scanning electron
microscopy experiments (Paper IV). TAT-N17Cdc42 on the other hand, had no effect on the
F-actin content. None of the TAT-proteins affected phagocytosis of IgG-opsonized yeast per
se (Paper IV), despite the fact that TAT-N17Cdc42 inhibited chemotaxis to a small extent
(20% reduction, Fig. 21, unpublished results).
55
non transduced
TAT-V12Cdc42
TAT-N17Cdc42
140
120
Migration
100
**
*
80
A
60
a
40
b
20
B
0
a
b
a/b
Figure 21. fMLP-induced chemotaxis of TAT-Cdc42 transduced HL60 cells.
Differentiated HL60 cells, non-transduced were transduced with constitutively active
(V12) and dominant negative (N17) TAT-Cdc42 proteins for 30 minutes. Three wells were
cut out in an agarose gel casted in a petri-dish. HL60 cells were applied to the mid-well,
fMLP in one well (A) and buffer in the other well (B). Cells were allowed to migrate
towards the chemoattractant for one hour. After fixation with PFA, the cells were stained
with Giemsa, and migration was evaluated in a light microscope, using an ocular
containing a ruler. Chemotaxis was evaluated as migration index (a/b), where a
represents the migration against a chemotactic gradient and b the spontaneous migration
of cells. Data are shown as mean ± SEM for migration (% of non transduced cells) of
three separate experiments run in triplicates.
The presence of an F-actin ring around the phagosome seemed to correlate with the lack of
CD63 positive granules (Paper IV) and was therefore subjected to further investigation. To
analyze translocation of CD63 positive granules, cells were loaded with Lysotracker® and the
movement of granules was recorded (Paper IV). Using quantitative image analysis, the
following scenario unravelled. In non-transduced cells, the ingested particle is located in a
phagosome surrounded by a transient ring of F-actin. Constitutively active Cdc42 induced the
formation of a thick F-actin ring affecting phago-lysosome maturation. The F-actin has to be
dissolved to allow fusion of granules with the phagosome. Cdc42 is also involved in the
actual translocation of granules to the phagosome, since dominantly negative Cdc42 resulted
in non-aggregated granules, randomly dispersed in the cell (Paper IV, and summarized in Fig.
22).
56
∗
Control
∗
∗
TAT-V12Cdc42
TAT-N17Cdc42
F-actin
CD63 positive granules
Figure 22. A schematic illustration on the effect of active/inactive Cdc42. In
control (non-transduced) cells the F-actin surrounding the phagosome is depolymerized
during phago-lysosome formation, allowing fusion of granules with the phagosomal
membrane. Transduction of cells with constitutively active (V12) form of Cdc42 induces
an accumulation of F-actin around the phagosome, impeding granule-phagosome fusion.
In cells transduced with dominant negative Cdc42 (N17), the appearance of the F-actin
ring around the phagosome resembles control cells, but translocation of granules to the
phagosome is impaired. The results summarized in this figure are presented in detail in
paper IV.
Transduction of HL60 cells with TAT-Pyk2
After optimizing the transduction method by using TAT-Cdc42, we were interested in
transducing the cells with TAT-Pyk2. By then, TAT-Pyk2 had successfully been transduced
into neutrophils (192). Differentiated HL60 cells were transduced with C-terminal part of
Pyk2, earlier shown to inhibit ROS production in neutrophils (192). It proved difficult to
transduce TAT-Pyk2 proteins into differentiated HL60 cells; probably due to the larger size of
TAT-Pyk2-fusion proteins compared TAT-Cdc42 proteins, and further optimization is
necessary. Nevertheless, protein transduction of HL60 cells with CT-Pyk2 had no effect on
the fMLP-mediated induction of β2 integrin affinity, nor on the β2- and fMLP-mediated
release of [3H]AA (data not shown); indicating that Pyk2 is not involved in these processes.
57
SUMMARY
Inhibition
Activation
Lxa, Aspirin
+
LTB4 -R
fMLP-R
Pyk2 P
ROS
ROS
AA
F-actin
IL-8-R
”Active”
β2 integrins
Lxa, Aspirin
LXA4 -R
Figure 23. The regulatory effect of pro- and anti- inflammatory mediators on
the β2 integrin-induced signalling in neutrophils. Prior to adhesion, integrin
receptors are activated by chemokines and chemotactic factors. The pro-inflammatory
chemoattractants fMLP and LTB4, mediate an increase in the lateral mobility and
clustering of β2 integrins. The affinity of β2 integrins, on the other hand, is mainly
increased by fMLP, and is not sensitive to treatment with the anti-inflammatory lipoxin
analogue (LXa). Also the β2 integrin-mediated tyrosine phosphorylation of Pyk2 is
insensitive to LXa treatment. Besides inducing phosphorylation of Pyk2, ligation of fMLPand β2 integrin-receptors (R) releases arachidonic acid (AA). Moreover, pre-incubation
of fMLP augments the β2-mediated release of AA. AA per se induces an increase in β2
integrin affinity and avidity and increases the amount of F-actin. Pre-incubation with the
anti-inflammatory lipoxin analogues (LXa) or treatment with aspirin, inhibits fMLPmediated activation of Pyk2, and the β2-mediated release of ROS. The data summarized in
this figure are presented in detail in Papers I-V, and as unpublished observations.
58
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
When investigating the regulatory effect of pro- and anti-inflammatory substances on the β2
integrin-mediated signalling in neutrophils, more questions were raised concerning:
i)
The activation state of the β2 integrins (i.e. affinity, avidity).
-
Does Pyk2 modulate the β2 integrin affinity/avidity?
-
Does Cdc42 mediate in the activation of β2 integrins?
-
What is the roll of small GTPases (e.g. Rac, Cdc42, and Rap1) in the
regulation of β2 integrin affinity?
ii)
The signalling capacity of β2 integrins (i.e. AA release and ROS production). More
specifically, the questions would be:
iii)
-
Does the anti-inflammatory lipoxin (LXa) affect the release of AA?
-
Does IL-8 induce release of AA?
-
How is the AA-release mediated in CAD patients?
-
Does AA per se induce the release of ROS?
-
Is the β2 integrin mediated release of ROS dependent of Pyk2 activation?
The signal transduction mediated by β2 integrins (i.e. Pyk2 phosphorylation). The
more specific questions would be:
-
Which tyrosine phosphorylation sites on Pyk2 are activated upon stimulation
with the pro-inflammatory chemoattractants LTB4, and fMLP?
-
What is the effect of LXa on these phosphorylation sites?
-
Does Pyk2 associate with β2 integrins prior or after stimulation with pro- and
anti-inflammatory substances? Does α-actinin/talin associate with Pyk2 in β2
integrin-stimulated HL60 cells? In other words, is Pyk2 a scaffolding protein
during β2 integrin-mediated signalling?
-
What effect do dominant negative- and constitutively active forms of Cdc42
have on the activation of Pyk2?
The questions are never-ending…
So, to my fellow scientists out there: I turn over the baton to you!
59
ACKNOWLEDGEMENTS
Many people have contributed in the making of this thesis, and I am grateful for all the
support I received along the way!
Detta är ett av de få tillfällen man har här i livet att verkligen tacka alla som har betytt något.
Så nu får ni stå ut med att det blir några sidor. Jag har ju haft turen att träffa så många trevliga
människor och lära känna så många nya vänner genom åren!
Jag vill speciellt tacka:
Min handledare, Eva Särndahl. Tack för allt stöd och all uppmuntran genom åren! Tack för att
du efter alla år (11) ser till att upprätthålla mitt intresse för forskning. Det är så mycket man kan
lära av dig: att vara en bra pedagog, att framgångsrikt skriva en forskningsplan och söka anslag,
och att inte ge upp i första taget! Jag har haft så kul när vi har rest tillsammans, trots förlorat
bagage och alla konstiga åkommor…
Tack för att du delat med dig av ditt liv och din familj, det har varit roligt att få se dina barn
växa upp. För att du alltid bryr dig och alltid ställer upp (ibland på bekostnad av din
familj…tack familjen!). Mest av allt vill jag tacka dig för att du har ”släppt mig lös” på lab och
att du (nästan) aldrig säger nej till mina vilda idéer! Du har under dessa år inte bara varit min
handledare utan också blivit en väldigt god vän...och vad mer kan man önska?
Olle Stendahl, min mentor och på den sista tiden också min biträdande-handledare.
Tack för att du engagerar dig, delar med dig av din stora kunskap, kommer med så bra
synpunkter och tack för att du alltid tar dig tid! Tack för att du är med oss i med och motgång...
och för att du blev nästan tusen gånger gladare än jag när manuset gick in till slut…
Pia Druid: Den bästa som finns! Tack för allt genom åren, inte bara all laborationsmässig hjälp
(som har varit super…”utan dig är vi intet”) utan också för allt ”skitsnack” och alla härliga
skratt. Tack för att du har brytt dig om och för att du ringt mig varje vecka under mina
barnlediga år!
Jane Wigren: Tack för att du såg till att labbet inte blev alltför ödsligt när Eva och Pia tillfälligt
”emigrerade”. Du är alltid så trevlig och hjälpsam och din korrekturläsning höjer engelskan till
nya nivåer. Vi syns vid kanalen i sommar (jag fika…du löpa…!)
Liselotte Ydrenius: Vad vi har haft kul genom åren! Hotten-Totten, tack för att din humor (om
än konstigare än min) och dina humörsvängningar. Det var min stora uppgift att retas med dig
tills du skrattade… jag lyckades för det mesta. Jag saknar det hasande ljudet av dina tofflor!
Martin Tinnerfelt Winberg, min ”lillebror” på labbet. Tack för allt skoj, för att du har stått ut
med alla ”tjuvnyp” och för att du gillar att kramas!
Sailesh Surapureddi, my sweetheart! Thanks for all the fun in the lab, for all the great meals
and for introducing me to “Pidgin English”.
Maria Lerm, för att du alltid hjälper till och alltid stöttar.
Alla våra studenter genom åren: Ida, Iida, Anna, Martin, Annelie, Calle, Malin, Pernilla,
Emma, Hanna och alla andra…tack för att ni bidragit till att höja stämningen på lab.
Några av mina rumskamrater genom åren: Jesper Svartz, Kristina Loinder och Tobias Strid.
60
Tänk vad mycket man får reda på i doktorandrummet! Tack för att jag fick störa er hela tiden.
Även om vetenskap kan vara kul att diskutera, så finns det ju andra saker här i livet som jag
hellre pratar om…som ni har märkt. Jesper, som också har varit NIR-doktorand, har jag haft
turen att få äta många lyxiga hotellfrukostar med i Lund. Vi har haft en massa skoj på alla kurser
och konferenser! Det finns bara en som du! På gränsen till för mycket…men sååå rolig!
Mina andra rumskamrater Johan Paulsson, Sofia Nyström och nyligen Sebastian Schultz.
Johan, ”the rising star over there”, du är inte bara trevlig, och smart…du är ju snygg också…jag
kommer att sjunga om dina biceps varje dag när du har åkt.
Sofia, en av mina ”medbrottslingar” på Cellbiologen sedan länge. Skönt att ha någon som vad
jag menar när jag pratar om ”gamla tider”. Jag kommer att sakna dina monologer och att hitta
dina nycklar och plånböcker överallt…Sebastian, tack för att du såg till att jag fick nya
”bevingade” kompisar på labbet.
”Strålforskningsgruppen” och ”Nyström gruppen”, nya som gamla: Unn Örtegren Kugelberg,
Karin Stenkula, Nabila Aboulaich, Anita Öst, Siri Fagerholm, Elisabeth Hallin, Hans
Thorn, Margareta Karlsson, Santiago Parpal, Anna Danielsson, Lilian Sauma, Nicklas
Franck, Fredrik Nyström, Johanna Gustavsson och alla nya som jag ännu inte har lärt
mig efternamnet på: Åsa, Iida, Cecilia och Gunnar. Tack för allt genom åren, för alla roliga
”spex” och fester. … arbetet är inte allt!
Nabila som bevisar att man kan vara supersmart och se bra ut samtidigt …och vara ödmjuk!
Anita, jag önskar att jag hade åtminstone hälften av din drivkraft. Och så är du så trevlig
dessutom. Framtida doktorander kan skratta sig lyckliga att få dig som handledare! Siri för att
du är alldeles för snäll! Elisabeth, som verkar vara redo att ”axla min mantel”, lika frispråkig
som jag (nästan), men skäller lite mer (men det kanske behövs) och jobbar desto mer.
”Hanse” som delade min skruvade humor och som jag hade så roligt med i forskarskolan …hur
var det nu med kavajen? Vi syns när du kommer tillbaka! Margareta, vår egen sångfågel, tack
för de här åren och hoppas att du orkar ta ton på en fest till! Santi för att du alltid avbröt dina
experiment för att hjälpa andra. Johanna, tack för alla djupa diskussioner om relationer och för
att du läste min avhandling.
Peter Strålfors, mannen vars åsikter jag oftast inte delar (det har nog inte undgått någon?!).
Tack för att du alltid har engagerat dig och brytt dig om hur det går för mig och för att du tar dig
tid! Jag kommer att sakna våra ”diskussioner”...Vem ska nu säga emot dig?
Gunilla Westermark, tack för alla sena diskussioner och för att du alltid delar med dig av din
gourmet mat! Jana Sponarova Jano, thanks for your friendship. I’m sorry that I am so bad at
Czech. Perhaps you can find someone to speak to at the party…?
Mats Söderström och Sven Hammarström. Mats, tack för all hjälp genom åren…datorer har
ett eget liv! Sven, tack för att du delar med dig av din stora kunskap och för att du tog dig tid att
läsa både manus och delar av avhandlingen.
Lärarna på Medicinsk Bilogi T3: Mats Lindahl, Tony Forslund och Margareta Lindroth.
Mats och Tony, det har varit kul att få ”bestämma lite” i terminsgruppen. Hoppas att ni hittar
något annat offer som vill läsa alla ”uppsatser”. Margareta, alltid så glad och så kunnig! Du är
bäst!...och söt i din nya frisyr…
”Gamlingarna” på HU: Carina Hellberg, Eva Grönroos och Ranghild Löfgren och Karin
Björnström. Tack Carina för att jag fick ta hand om ”jätte-bebisen” Tulle. ”Evis”, du och jag
tillsammans blir nästan för mycket.
61
De övriga på Cellbiologen, Mikrobiologen, och Farmakologen: Lotta Ericsson, Anna Berg,
Cecilia Trinks, Peter Påhlsson Maria Lerm, ”flödesexperten” Tony Forslund, Torbjörn
Bengtsson, Maria Forsberg, Åsa Holm, Charlotte Immerstrand, Birgitta Rasmusson och
alla ni andra.
Anna, nu när jag kommer att ha all tid i världen så måste vi faktiskt se till att träffas!
Alla mina vänner från Biomedicinska forskarskolan: Charlotte Anna, Petra, Karin, Hans,
Johan, Jacob, Ylva, Marcus, och så naturligtvis Torbjörn och Lillemor! Tack för ett roligt
år!...jag fick ”kexet” till slut!
Mina vänner från NIR: speciellt Maria, Lisa, Veronica och Cecilia. Vi har haft så kul när vi
har bott på Patienthotellet i Lund! Tack till Mark Hickery och Maria Olofsson för ett superbra
jobb!
All the people at the Ludwig Institute for Cancer Research in Uppsala: the “Boss” Calle Heldin,
Ivan, Inga, my great friends Ninna, Jill, Sofia and Eva G. The rest of “the gang” Tobias, Olli,
Johan, my room-mate Anders, Ann-Sofie, Andree, and of course cykelfixaren Ulf. Thank you
for taking care of me and for all the fun during the late lab-nights. My supervisor Ivan Dikic,
thank you for introducing me to the realities of science and the “importance” of being focused
and dedicated…
The people “over there” in Indianapolis: Fred Pavalko, Sarah Sawyer and all the rest. Fred,
thank you for taking such good care of me during my stay! Your never failing enthusiasm,
encouragement and kindness will not be forgotten. I had a great time! Thank you for giving me a
ride to the lab every morning. Sarah, thank you for being such a good friend and for helping me
spending my money at great restaurants and in book shops! All the rest of the people at the
Indiana University School of Medicine who I had the fortune to meet…I miss you all!
Patrik Lindblom, utan din hjälp hade vi inte kunnat få iväg några artiklar! Och tack för all
super-god mat genom åren!
Förutom forskning, så har man ju faktiskt andra intressen här i livet. Därför finns det
en hel del människor som jag vill tacka!
Unn och Fredrik Kugelberg. Tack för att ni är så goda vänner. Ni är för snälla för ert eget
bästa! Tack för alla goda middagar, viner och framför allt alla icke vetenskapliga diskussioner
genom åren och för allt stöd. Nu när förkylningsperioden är över så kanske jag kan få krama på
Inger lite mer! Hoppas att ni slipper flytta åt oss en femte gång…
Karin Stenkula och Johan Tengelin. Tack för all vänskap. Alla goda middagar (ett
återkommande tema!) och alla skratt. Ihop med yrvädret Karin kan man inte annat än skratta!
Karin, tack för att du har delat mitt ”livsfarliga” hästintresse genom åren… Synd att ni har
flyttat till Örebro…men vem vet vi kanske flyttar efter?!
Charlotte och Björn Immerstrand. Charlotte, den mest positiva människan jag någonsin
träffat!...förutom din man Björn kanske. Det är alltid så trevligt att umgås med er och era barn.
Vi ser fram emot att plaska i poolen i sommar också!
Helena Negga. Det är sällan som man träffar någon som ger så mycket av sig själv utan att
kräva något tillbaka, men du är precis en sådan människa! Tack för allt ditt stöd, för dina kloka
råd och din inställning till livet. Nu kanske vi kan träffas mer! Micke, tack för din vänskap och
för att du pratar träning med min man…så att jag slipper.
62
Christel Winell för allt roligt vi hade på labbet och för att jag fick vara en del av din familj
under en ganska lång tid…
Josefin Axelsson (numera Perman), ”biologen” som övergav mig till förmån till läkarlinjen. Det
var ju så tråkigt att du slutade, men idag är jag glad att vi har en egen ”familjeläkare”. Tack för
alla våra låånga telefonsamtal genom åren, för allt fikande och shoppande. Det var kul att titta på
tv (läs ER) tillsammans per telefon… Hoppas att jag får träffa guldklimpen snart!
”Mahjong-gänget” : Camilla Wohlgemuth, Annika Silvervarg och Kristina Pieslinger. Tack
för alla Mahjong dagar, med chips, dipp, en massa prat och lite spelande…tänk att jag inte har
vunnit en hel omgång en enda gång, trots att vi har spelat i sex (?!) år! Camilla, tack för att du
har varit min vän sedan den dagen jag knackade på hemma på Lövudden och ville låna
böcker…Man blir så glad av att träffa dig!
Alla våra trevliga grannar. Tack för att ni ser till att vi inte vill flytta ifrån Berg!
Ulrika och Peter Bergman, tack för att ni hjälper oss så mycket med allt! Passar våra barn och
bjuder på god mat. Jag uppskattar det! Tack Ulrika för att du utan att tveka skjutsade in mig till
jobbet när jag behövde referenser och för att du släpar ut mig på promenader så att jag får ”röra
på fläsket”.
Karin och Magnus Seger, våra ”jobbiga” grannar tvärs över som alltid fixar på huset, i
trädgården eller är ute och motionerar…suck! Karin tack för alla kaffepauser, goda kakor,
middagar och allt ”skitsnack”…jag behövde det när jag skrev! Vilken tur att du håller mig
uppdaterad på allt som händer i Berg! Tack för att ni inte har ”gjort er av med” våra katter
ännu…
Sist, men inte minst så måste jag få tacka mina nära och kära.
Mina svärföräldrar Inger och Lars, tack för att ni har hjälpt oss så mycket genom åren.
My family in the Czech republic. I hope to see you soon!
Min familj som ibland kan likna ”the Fockers”…
Min mamma Rumenka som alltid sätter sig i bilen och kommer om jag behöver det. Tack
mamma för att du alltid stöttar mig i det jag vill göra. Jag hoppas att jag kan uppfostra mina barn
lika bra som du har gjort! Stig, som är bra på allt! Vår egen handy-man som fick en massa barn
på köpet när han gifte in sig i vår familj.
Min syster Petra och Andreas, familjens ”Barbie och Ken ” (lika snygga men mycket
smartare). Petra, …lika rättfram som jag, men inte riktigt lika diplomatisk…en konstnärsjäl
som är bra på det mesta. Jag är så glad att du finns! Vi har haft mycket kul genom åren. Tack för
alla sena nätter på msn… Puss till mosters Isac!
63
Min lillebror Daniel (inte så liten nu för tiden). En duktig musiker, designer, helt enkelt skitbra
på att flyga modellflygplan…och att ragga tjejer på nätet. Jag är stolt över dig!
Mina barn, Ella och Alexander. För all er villkorslösa kärlek!
Min man, Greger. Att skriva avhandling med två små barn är stundtals väldigt jobbigt och då
behöver man allt stöd man kan få. Greger, tack för att du har funnits vid min sida i alla dessa år.
Du är den mest kärleksfulle och omtänksamme man och pappa man kan tänka sig…om än
stundtals lite tankspridd. Vilken tur att jag inte gav upp fast du fattade så trögt! Tack för all din
kärlek. Jag älskar dig!
The research presented in this thesis was supported by the following grants:
•
Ph.D. student fellowship from the Network for Inflammation research (NIR) funded by the Swedish
foundation for Strategic Research (SSF)
•
Fellowship from the Swedish Heart & Lung Foundation
•
Swedish Research Council
•
King Gustaf V Memorial Foundation
•
Swedish Medical Society
•
Medical County Council of Östergötland
•
Lars Hierta Foundation
64
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