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Mechanisms involved in macrophage
phagocytosis of apoptotic cells
Anna Nilsson
Department of Integrative Medical Biology
Section for Histology and Cell Biology Umeå University, Umeå, Sweden
2009
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Cover illustration: Confocal microscopic image of murine resident peritoneal macrophages, stained for F-actin (green), CMFDA labeled apoptotic thymocytes (pink), and nuclear DAPI staining (blue).
© Anna Nilsson 2009 Umeå University Medical Dissertations New Series No. 1307 ISSN 0346-6612 ISBN 978-91-7264-890-6 Printed at Print & Media, Umeå University, Umeå, Sweden
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To Stefan and Hjalmar
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TABLE OF CONTENTS
ABBREVIATIONS 6
ABSTRACT 7
LIST OF ORIGINAL PAPERS 8
INTRODUCTION 9
Apoptosis..................................................................................................................................................9
Elimination of apoptotic cells - an overview..........................................................................................10
“Find me signals”....................................................................................................................................11
“Eat me signals”......................................................................................................................................12
The calreticulin/LRP1 interaction...........................................................................................12
“Don’t eat me signals”............................................................................................................................14
The CD47/SIRPα interaction..................................................................................................14
Engulfment and degradation...................................................................................................................17
Immunological consequences of defective apoptotic clearance.............................................................17
Senescent erythrocytes............................................................................................................................18
Macrophages...........................................................................................................................................19
Macrophage subpopulations....................................................................................................20
Dendritic cells.........................................................................................................................................20
AIMS 23
RESULTS 24
LRP1 - a prophagocytic receptor...........................................................................................................24
CRT on the apoptotic cells is involved in their uptake...........................................................24
LRP1 is the CRT-receptor mediating phagocytosis................................................................24
The expression level of LRP1 can be modulated by glucocorticoids and modulate macro-
phage phagocytic capacity......................................................................................................25
Changed distribution and expression of CD47 and CRTon apoptotic cells...........................................26
CRT expression is upregulated and redistributed an apoptotic cells.......................................26
Changes to CD47 on the surface of apoptotic cells differ between cell types........................26
CD47 on apoptotic murine thymocytes facilitates their uptake by mediating binding of..........
apoptotic cells to the macrophages..........................................................................................26
Redistribution of CRT and CD47 into certain areas on the apoptotic cell surface.................27
Role of CD47 in the phagocytosis of experimentally senescent erythrocytes........................................27
CD47 does not affect phagocytosis of ionomycin-treated PS+ Ca2+ RBCs in vitro................28
In vivo clearance of experimentally senescent erythrocytes...................................................28
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DISCUSSION 30
LRP1/CRT interaction and apoptotic cell removal.................................................................................30
CD47 on apoptotic cells..........................................................................................................................32
Clearance of experimentally senescent erythrocytes..............................................................................34
CONCLUSIONS 37
ACKNOWLEDGEMENTS 38
REFERENCES 40
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ABBREVIATIONS
SIRPα signal regulatory protein alpha
CRT calreticulin
LRP1 low density lipoprotein receptor-related protein 1
RAP receptor-associated protein
PS phosphatidylserine
RBC red blood cells
BMM bone marrow-derived macrophages
RPM resident peritoneal macrophages
GC glucocorticoid
GCR glucocorticoid receptor
cGCR cytosolic glucocorticoid receptor
Dex dexamethasone
DC dendritic cell
ITIM immunoreceptor tyrosine-based inhibitory motifs
GM-CSF granulocyte-macrophage colony-stimulating factor
M-CSF macrophage colony-stimulating factor
MEF mouse embryonic fibroblast
MZ marginal zone
TSP thrombospondin
Wt wild-type
Ca2+-RBCs ionomycin-treated experimentally senescent PS+ erythrocytes
AIHA autoimmune hemolytic anemia
MHC major histocompatibility complex
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ABSTRACT
Efficient removal of apoptotic cells is critical for development, tissue remodelling, maintenance of homeostasis, and response to injury. Phagocytosis of apoptotic cells is mediated by many phagocytic receptors, soluble bridging molecules, and pro-phagocytic ligands on the surface of apoptotic cells. Macrophage phagocytosis in general is controlled by stimulatory and inhibitory mechanisms. An example of the latter mechanism is that mediated by the cell surface glycoprotein CD47, which by binding to the inhibitory receptor Signal Regulatory Protein alpha (SIRPα) on macrophages, is known to inhibit phagocytosis of viable host cells. The studies of the present thesis aimed at investigating possible changes to CD47 on apoptotic cells, which could influence their elimination by macrophages. The endoplasmatic protein calreticulin (CRT), in conjunction with Low density lipoprotein Receptor-related Protein 1 (LRP1) on the phagocyte, can act as a receptor for collectin family members and mediate uptake of apoptotic cells. However, CRT itself was found to also be expressed on the surface of many viable cell types, and the CRT expression increased on apoptotic cells. By using antibodies to LRP1 or receptor-associated protein (RAP), an antagonist blocking LRP1 ligand binding, we found that CRT on target cells could interact in trans with LRP1 on a phagocyte and stimulate phagocytosis. CD47 on the target cell inhibited LRP1-mediated phagocytosis of viable cells (e.g. lymphocytes or erythtocytes), but not that of apoptotic cells. The inability of CD47 on apoptotic cells to inhibit LRP1-mediated phagocytosis could be explained in two ways: 1) Some apoptotic cell types (fibroblasts and neutrophils, but not Jurkat T cells) lost CD47 from the cell surface, or 2) CD47 is evenly distributed on the surface of viable cells, while it was redistributed into patches on apoptotic cells, segregated away from areas of the plasma membrane where the pro-phagocytic ligands CRT and phoaphatidylserine (PS) were concentrated. Apoptotic murine thymocytes also showed a patched distribution of CD47, but no significant loss of the receptor. However, both PS-independent and PS-dependent macrophage phagocytosis of apoptotic CD47-/- thymocytes was less efficient than uptake of apoptotic wild-type (wt) thymocytes. This contradictory finding was explained by the fact that CD47 on apoptotic thymocytes did no longer inhibit phagocytosis, but rather mediated binding of the apoptotic cell to the macrophage. These effects could in part be dependent on the apoptotic cell type, since uptake of experimentally senescent PS+ wt or CD47-/- erythrocytes by macrophage in vitro, or by dendritic cells (DC) in vivo, were the same. In vivo, PS+ erythrocytes were predominantly trapped by marginal zone macrophages and by CD8+ CD207+ DCs in the splenic marginal zone. DCs which had taken up PS+ erythrocytes showed a slight increase in expression levels of CD40, CD86 and MHC class II. These findings suggest that PS+ erythrocytes may be recognized by splenic macrophages and DCs in ways similar to that reported for apoptotic T cells. Uptake of senescent erythrocytes by DCs may serve as an important mechanism to maintain self-tolerance to erythrocyte antigens, and defects in this function may facilitate development of AIHA. Glucocorticoids are used to treat inflammatory conditions and can enhance macrophage uptake of apoptotic cells. We found that the glucocorticoid dexamethasone time- and dose-dependently stimulated macrophage cell surface LRP1 expression. Dexamethasone-stimulated macrophages also showed enhanced phagocytosis of apoptotic thymocytes and unopsonized viable CD47-/- erythrocytes. In summary, LRP1 can mediate phagocytosis of both viable and apoptotic cells by binding CRT on the target cell. Macrophage expression of LRP1 is increased by glucocorticoids, which could be one explanation for the anti-inflammatory role of glucocorticoids. While CD47 on viable cells efficiently inhibits phagocytosis in macrophages, CD47 on apoptotic cells does not and can sometimes even promote their removal.
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LIST OF ORIGINAL PAPERS
The papers will be referred to in the text by their roman numerals.
I. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Gardai, S.J., McPhillips, K.A., Frasch, S.C., Janssen, W.J., Starefeldt, A., Murphy-Ullrich,J.E., Bratton,D.B., Oldenborg, P.-A., Michalak, M., and Henson, P.M. Cell, 2005; 123:321-334.
II. Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids.
Nilsson, A., Vesterlund, L., Oldenborg, P.-A. Manuscript
III. CD47 promotes both phosphatidylserine-independent and phosphatidylserine-dependent phagocytosis of apoptotic murine thymocytes by non-activated macrophages. Nilsson, A. and Oldenborg, P.-A. Biochem. Biophys. Res. Commun, 2009; 387:58-63.
IV. Role of CD47 in regulating macrophage and dendritic cell uptake of calcium-induced experimentally senescent erythrocytes in vitro and in vivo. Nilsson, A.*, Larsson, A.*, Olsson, M., Oldenborg, P.-A. Manuscript *Equal contribution
All published papers are reproduced with the permission of the copyright holders
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INTRODUCTION
Apoptosis Apoptosis is a physiological process and a type of programmed cell death which is important
for embryologic development, maintenance of homeostasis and elimination of damaged cells.
This is a controlled, energy dependent process and involves a number of different
morphological changes. These changes are a result of the action of efficient cysteinyl
aspartate-specific proteases called caspases (7), and includes reduced cell volume, chromatin
condensation, nuclear fragmentation, and membrane blebbing (8). Apoptotic death can be
triggered by a wide variety of different stimuli, which induce either the extrinsic or the
intrinsic apoptotic pathways. The extrinsic pathway is induced by the activation of pro-
apoptotic receptors on the cell surface. Specific molecules such as Apo2L/TRAIL and Fas-
Ligand, which are known as pro-apoptotic ligands, bind to the death receptors (DR)4/DR5
and Fas, respectively, and thereby induce activation of apoptosis (14). In contrast to the
extrinsic pathway, the intrinsic pathway is initiated from within the cell in response to stress
signals, such as DNA damage, hypoxia, or the loss of survival signals (15,16). Both pathways
are however interconnected with other signalling proteins, such as NK-κB and p53-MDM2,
and also converge at the level of effector caspases (7). Caspases are proteolytic enzymes that
mediate apoptosis by cleavage of a number of different substrates that are vital for cell
survival (21,22). In response to apoptotic stimuli, initiator caspases are activated by pro-
apoptotic signals, where different stimuli trigger different initiator caspases, which in turn
start an activation cascade of effector caspases, responsible for the cleavage of vital cellular
components and thereby carrying out apoptosis (22). Under normal conditions, regulation of
caspase activity is controlled by the action of inhibitors of apoptosis (IAP) proteins (7).
Apart from morphological changes during apoptosis, changes on the apoptotic cell surface
also occur. These changes are mainly important to stimulate efficient removal of apoptotic
cells by phagocytosis. However, apoptosis is a dynamic process, during which cell surface
molecules are continuously changing. Phosphatidylserine (PS), a phospholipid that is
normally present on the inner leaflet of the plasma membrane, flips to the outer leaflet early in
the apoptotic process. Increased PS expression has been found to stimulate phagocytosis (24).
As a result of both oxidative and non-oxidative stress-induced apoptosis, expression of
oxidized PS, in conjunction with the non-oxidized PS, serves as an important phagocytosis-
stimulating signal (25). Other cell surface changes are alterations in the pattern of
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glycosylation of glycoproteins and glycolipids (24,26), changed expression levels of specific
molecules, and non-specific changes such as surface charge possibly due to changes in
glycosyl groups (27,28). Alterations in sugar chains, surface charge and oxidation result in the
generation of sites resembling oxidised lipoprotein particles, thrombospondin (TSP) binding
sites, sites capable of binding lectins or the complement proteins C1q and C3b, as well as
various collectin-binding sites. These surface alterations, resulting in new binding sites for
receptors, have important implications for the removal of the apoptotic cell (29-31).
Elimination of apoptotic cells – an overview The efficiency whereby apoptotic cells are removed is extremely high. It has even been
suggested that detection of apoptotic cells in vivo indicates a possibility of defective clearance
mechanisms, or a large overload of apoptotic cells. Elimination is accomplished by
professional phagocytes like macrophages and dendritic cells (DCs), but also by non-
professional phagocytes such as fibroblasts, epithelial and stromal cells to name a few cell
types (27) (Fig.1). To recruit professional phagocytes, that are not always located in
immediate proximity, apoptotic cells have been found to secrete soluble molecules that act as
chemotactic factors, “find me” signals, to phagocytes. Following recruitment of phagocytes,
the elimination of apoptotic cells consists of two central elements: 1) the apoptotic cell has to
be tethered to the phagocyte, and 2) induction of phagocytosis-stimulating signals upon cell-
cell contact. These interactions between the apoptotic cell and the phagocyte are key events
for proper removal (32). Generally it is suggested that these interactions induce phagocytosis
stimulatory (“eat me”) or inhibitory (“don’t eat me”) signals and that the relative contribution
of these signals determines whether the cell should be eliminated or not. Viable and apoptotic
cells have different expression patterns of cell surface structures, where apoptotic cells in
contrast to viable cells preferentially induce stimulating signals resulting in their elimination
(27,33). Internalization is stimulated by activation of Rac-1 and Cdc42, both members of the
Rho family of GTPases, and they affect cytoskeletal organization (32). Cytoskeletal
rearrangement enables the phagocyte to enclose the apoptotic cell into a phagosome where
degradation occurs (34). Degradation of apoptotic cells by DCs plays an important role in the
maintenance of the peripheral tolerance (35).
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Eat me
a) Soluble factors released from the apoptotic cell induces find me signals.
Phagocyte
b) Induction of eat me and don’t eat me signals upon contact.
Apoptotic cell
Phagocyte
Apoptotic cell
Phagocyte
c) A majority of eat me signals activates phagocytosis
Don’t eat me
d) Engulfment
Apoptotic cell
a) Soluble factors released from the apoptotic cell induces find me signals.
Phagocyte
b) Induction of eat me and don’t eat me signals upon contact.
Apoptotic cell
Phagocyte
Apoptotic cell
Phagocyte
Apoptotic cell
Phagocyte
Apoptotic cell
Phagocyte
c) A majority of eat me signals activates phagocytosis
Don’t eat meDon’t eat me
d) Engulfment
Apoptotic cell
Apoptotic cell
Figure 1. Phagocytosis of apoptotic cells a) Apoptotic cells release soluble factors that act as chemoattractants for phagocytes. b) As the phagocyte establishes a contact with the apoptotic cell, numerous interactions between their receptors and ligands occur. These interactions might induce phagocytic stimulatory (“eat me”) signals, or inhibitory (“don’t eat me”) signals. c) It is thought that it is the balance between these two signals that determine whether phagocytosis should occur. The interaction with an apoptotic cell, in contrast to a viable cell, induces a majority of “eat me” signalling, resulting in d) engulfment into a phagosome. Adopted figure from Molecular Cell, Vol 14, 277-287, May 7, 2004. Lauber K. et. Al. Clearance of apoptotic cells: Getting rid of the corpses
“Find me signals” A proposed factor in the recruitment of phagocytes to the location of apoptotic cells, is the
production of lysophosphatidylcholine (LPC), the release of which is triggered in apoptotic
cells by caspase-3 mediated cleavage and activation of the calcium independent
phospholipase A2 (iPLA2) (36). Upon secretion, it induces macrophage migration, presumably
by binding to the G protein coupled receptor G2A (37). Endothelial monocyte-activating
polypeptide ІІ (EMAP ІІ) is the product of another cleavage occurring during apoptosis and
serves as a ligand for the interleukin-8 (IL-8) receptor (38). IL-8 is an important mediator of
leukocyte infiltration into inflamed tissues, which is mimicked by EMAP ІІ. Recently
shingosine-1-phosphate (S1P), released by apoptotic cells (39), was found to mediate
monocyte/macrophage migration in vitro (40).
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“Eat me” signals Recognition of the apoptotic cell is mainly accomplished by alterations on the apoptotic cell
surface, which upon contact with phagocytes induce “eat me” signals. The best characterized
cell surface change is exposure of PS, which is translocated to the cell surface in the early
phase of apoptosis (41). Several phagocytic receptors are attracted to PS through indirect
interaction mediated by bridging molecules. For example, integrins (αvβ3 and αvβ5) bind PS
via the secreted protein milk fat globule-EGF factor 8 (MFG-E8) (42,43), and Mer receptor
tyrosine kinase binds via the soluble protein growth arrest-specific 6 (Gas6) (44). Bridging
molecules are also involved in recognizing alterations of sugars and/or lipids on the apoptotic
cell surface. These include members of the collectin-family members surfactant proteins A
and D (SP-A and SP-D), mannose-binding lectin, and the collectin-like first component of the
classical complement cascade C1q (31,45). It has been speculated whether there might be a
specific PS receptor on the phagocyte that directly binds PS. Three receptors have recently
been suggested to interact directly with PS and to induce uptake of apoptotic cells: Tim4 (T-
cell immunoglobulin- and mucin-domain-containing molecule), BAI1 (brain-specific
angiogenesis inhibitor 1) (46,47), and Stabilin-2 (48).
There are also other phagocyte receptors that interact directly with surface structures on the
apoptotic cell. Scavenger receptors such as CD68, scavenger receptor A (SR-A) (49), and
lectin-like oxidized LDL particle receptor 1 (LOX-1) (50), all binds to oxLDL-like sites (33).
CD36 binds to TSP-1 binding sites (33), and the lipopolysaccharide receptor CD14 binds to
altered ICAM3 on the apoptotic cell surface, without any inflammatory consequences (51).
The calreticulin/LRP1 interaction
Calreticulin (CRT) is a Ca2+-binding endoplasmatic protein and a molecular chaperone. As
such, it is a multifunctional protein that mainly functions in the endoplasmic reticulum (ER).
However, by still unknown mechanisms, CRT is transported to the plasma membrane and is
detectable on the cell surface. As a cell surface protein CRT has been implicated in antigen
presentation and complement activation, immunogenicity of cancer cell death, wound healing,
thrombospondin signalling, and clearance of apoptotic cells (52). The role of CRT in
clearance of apoptotic cells involves its association with low density lipoprotein Receptor-
related Protein 1 (LRP1) on the phagocyte, where it functions as a binding site for the
collagenous tails of collectin-family members (e.g. mannose binding lectin (MBL), SP-A and
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Figure 2. The CRT/LRP1 cell surface complex Cis interaction between CRT and LRP1on the phagocyte function as a binding site for the collagenous tails of collectin-family members. C1q and SP-A are members of the collectin-family that have the ability to bind to apoptoic cells and thereby facilitate their elimination. Reprinted by permission from Macmillan Publishers Ltd: Nat Rev Mol Cell Biol (1), copyright (2001)
SP-D). C1q, the first component of the classical complement pathway, is related to this family
because of its structural similarities. Binding of C1q to apoptotic cells has previously been
observed. Opsonisation of apoptotic cells by C1q enables detection and subsequent
phagocytosis upon interaction with the CRT/LRP1-complex on the phagocyte. Therefore CRT
has a role in phagocytosis of apoptotic cells acting in a cis mode with LRP1 (Fig. 2) (31,45).
The LRP1 receptor is a member of the low-density lipoprotein (LDL) receptor superfamily
together with LRP1b, megalin/LRP2, LDL receptor, very low-density lipoprotein receptor
(VLDL receptor), MEGF7/LRP4, and LRP8/apolipoprotein E (apoE) receptor 2. Due to a
post-translational cleavage, LRP1 is composed of an extracellular α-chain and a
transmembrane β-chain that together form a
non-covalent heterodimer (53). It is a large
multifunctional receptor found in most
tissues and is involved in two major
physiological processes; endocytosis, and
regulation of intracellular signalling
pathways. As many as 40 different ligands
can interact with LRP1, and the resulting
endocytosis of these ligands therefore
regulates the levels of several molecules by
facilitating their delivery to lysosomes for
subsequent degradation. Ligands range
from lipoproteins, extracellular matrix
proteins, protease/protease-inhibitor
complexes, and viruses, to cytokines and
growth factors (54). The regulatory
functions of LRP1 include controlling
vascular smooth muscle cell proliferation
(55) and regulating blood-brain barrier
permeability (56). Moreover, LRP1 on
neurons regulate calcium signalling, an
important second messenger for neuronal
response to excitatory neurotransmitters
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such as glutamate (57), which play a role in neuronal degradation (58). LRP1 has also been
suggested to have a role as a regulator of the inflammatory response in the lung (59). The
exact mechanisms behind these signalling pathways are not fully understood. The
cytoplamatic tail of LRP1 contains several potential signaling motifs: two NPxY motifs, an
YXXL motif, and two dileucine motifs. The YXXL motif might be most dominant in
regulating endocytosis signaling, while the two NPxY motifs are capable of interacting with
cytoplasmic adaptor proteins and scaffold proteins (54). A large number of intracellular
proteins interact with the cytoplasmic tail of LRP, thus connecting it to several different
signaling pathways. Recently, a new mode of signaling was ascribed to LRP1, which involves
cleavage of the receptor within the plane of the membrane segment. This resulting 12kDa
intracellular domain (LRP-ICD) has the ability to translocate within the cell and elicit
physiological responses (60).
“Don’t eat me” signals Cells might constantly be targeted for removal and actually have to prove their viability in
order not to be eliminated (33). Therefore there are also inhibitory interactions between cells
and phagocytes that induce “don’t eat me” signals. CD31 on a viable cell interact
homotypically with CD31 on a potential phagocyte, resulting in its detachment and
consequently no engulfment will take place. The inability of CD31 on apoptotic cells to do
the same enables their ingestion (61). Furthermore the interaction between CD47 on host cells
and SIRPα on macrophages or DCs induces an inhibitory signal, preventing phagocytosis of
the viable cell, making CD47 a possible marker of self (2-4,62). In the same way as “eat me”-
inducing molecules are upregulated, or structurally altered on the apoptotic cell surface,
“don’t eat me” molecules are altered in ways that disrupt their ability to inhibit phagocytosis
(63). In this way, “eat me” signals could be allowed to be present on viable cells, together
with “don’t eat” me signals, since the balance between these signals will determine whether a
cell should to be eliminated or not (64,65). On an apoptotic cell, changes occur that increases
the amount of “eat me” signals and reduces the “don’t eat me” signals, which will facilitate
apoptotic cell uptake by the phagocyte.
The CD47/SIRPα interaction
CD47, or integrin-associated protein (IAP), is a ubiquitously expressed transmembrane cell
surface glycoprotein and a member of the immunoglobulin (Ig) superfamily. It was first
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Table 1. Biological functions of CD47
Ligand/associated protein Function Reference SIRPα Marker of self, inhibits phagocytosis (3,4)
SIRPα Regulaion of cell migration (5)
SIRPα Macrophage fusion and osteoclast formation (6)
α-CD47 mAbs
SIRPα/γ
TSP-1
FAS/CD95-association
Induction of apoptosis
(9-13)
SIRPα, TSP-1 T cell activation/inhibition Treg formation (17-19)
β1, β2, β3, β5 integrin association Regulation of integrin –mediated activation (20)
TSP-1 Inhibits NO-dependent production of cGMP (23)
identified in association with the integrin αvβ3, hence it alternative name IAP. Structurally
CD47 has a IgV-like extracellular domain, five putative membrane-spanning segments and a
short cytoplasmic tail, where it is the Ig domain that is important for the interaction with
CD47 ligands (20). Four splicing alternatives have been found in vivo for the cytoplasmatic
tail (66), where the second-shortest, form 2, is the predominant one. The different biological
outcomes following the interaction between CD47 and its different ligands are summarized in
table 1.
SIRPα is a plasma-membrane protein (also known as SHPS-1, CD172a, BIT, MFR and P84)
(67), known to interact with CD47 (2). It was the first of the three members of the SIRP-
family to be identified (68). Structurally, all three receptors, SIRPα, SIRPβ and SIRPγ, have
an extracellular region consisting of three Ig-like domains, where there are two IgC-like
domains and one IgV-like domain. However, the cytoplasmatic regions of the SIRP family-
members are different. The cytoplasmatic region of SIRPα has four immunoreceptor tyrosine-
based inhibitory motifs (ITIMs), whereas SIRPβ has a very short cytoplasmatic tail with no
cytosolic signalling motifs. Instead, the transmembrane region contains a positively charged
lysine residue to which the immunoreceptor tyrosine-based activating motif (ITAM)-carrying
adaptor protein DAP12 can bind. SIRPγ has no recognizable signalling motif and is therefore
unlikely to generate intracellular signals (68,69). SIRPα and SIRPβ are present on myeloid
cells, and SIRPα has in addition also been found on neuronal cells. SIRPγ expression can be
found on T cells and activated NK cells (69,70). CD47 interacts with both SIRPα and SIRPγ,
but only weakly with SIRPβ. The interaction with SIRPα induces a number of different
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Figure 3. The CD47/SIRPα-interaction Interaction between CD47 and SIRPα can result in a bidirectional signaling. Contact is mediated between the Ig-like domain of CD47 and the N-terminal IgV domain of SIRPα. Upon interaction, tyrosine phosphorylation of the cytoplasmic domain of SIRPα occurs, enabling binding of protein tyrosine phosphatases SHP-1 and SHP-2. SHP-1 negatively regulates cellular functions whereas SHP-2 many times has a positive effect (2). Reprinted from Trends Cell Biol. Feb. 19(2), Matozaki T., Murata Y., Okazawa H., Ohnishi H., Functions and molecular mechanisms of the CD47-SIRPα signaling pathway, 72-80 (2009), with permission from Elsevier.
biological responses. Upon ligand binding, the ITIMs of SIRPα is phosphorylated and
mediate recruitment and activation of the tyrosine phosphatases SHP-1 and SHP-2, which in
turn dephosphorylate specific protein targets, thereby regulating cellular functions. This is
often a negative regulation, where the best documented role of SIRPα is the inhibition of
phagocytosis upon binding to CD47 on host cells (Fig. 3). Besides this, SIRPα is also
important for controlling myeloid cell migration (5), DC and T cell activation (18,71,72), and
formation of multinucleated cells in vitro and in vivo (6,73). The exact expression levels of
both SIRPα (74) and CD47 (75) has been found to affect the outcome of the SIRPα/CD47
interaction.
An altered expression level of CD47 may play a role in tumour development. Tumour cells
express stress-ligands on the cell surface, which may target them for NK cell and phagocyte
recognition. Earlier studies demonstrated that CD47 was identical to the cancer antigen OV-3,
which is upregulated on ovarian carcinoma cells (20). In addition, recent observations showed
an upregulation of CD47 on leukemia cells enabling them to avoid phagocytosis (76).
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Engulfment and degradation In addition to the exposure of “eat me” signals on apoptotic cells, there is also a requirement
for redistribution of the responsible molecules into clustered patches in the process of
elimination. Oligomerization of low affinity interactions results in an optimal stimulation
(33). Therefore important players in the engulfment process are clustered together, forming an
engulfment synapse, optimizing the interactions between the apoptotic cell and the phagocyte
(27,33). Internalization is a process that profoundly depends on the phagocytic receptor
involved (33). It has been suggested that internalization of the apoptotic cell differs from the
“zipper” mechanism seen in Fcγ receptor-mediated uptake in the way that the process appears
to be more similar to macropinocytosis, with formation of a spacious phagosome, resulting in
uptake of surrounding fluid (77,78). However, another study showed that apoptotic cell
internalization is more similar to the “zipper”-like phagocytosis (79). Internalization is actin-
dependent and the signalling mediators in the two proposed pathways resulting in cytoskeletal
rearrangement all converge at Rac-1. One pathway involves ELMO, CrkII, DOCK180 and
Rac-1 and the other pathway involves LRP-1, Gulp, ABCA1 and Rac-1 (80,81). In addition,
Rac-1 activates RhoA, known to inhibit uptake (81), which may explain why phagocytes lose
the ability to ingest a second portion of apoptotic cells (82). Engulfment of the apoptotic cell
generates a phagosome that matures by a series of fusion and fission events culminating in the
mature phagolysosome (83). Phagosome maturation as well as engulfment is in part regulated
by the balance between Rho/Rac (33).
Immunological consequences of defective apoptotic clearance In contrast to pathogen elimination, apoptotic cell clearance does not result in an
inflammatory response. This is due to more than a passive avoidance; instead apoptotic cells
are actively immunosuppressive. Interaction between the apoptotic cell and the phagocyte
induces suppression of the pro-inflammatory cytokines TNF-α, GM-CSF, IL-12, IL-1β and
IL-18, and stimulates the release of the anti-inflammatory cytokines IL-10 and TGF-β (84).
Suppression of inflammatory responsiveness is primarily initiated at the level of specific
transcriptional initiation (85). In addition constituents from apoptotic cells are recycled to the
membrane of the phagocyte, where the presentation of these antigens to T cells is a central
event in the induction and the maintenance of peripheral immune tolerance (86,87).
Interestingly, a study showed that transfusion of apoptotic splenocytes from the donor strain
prior to transplantation prolonged the survival of heart allografts in a rat model (88).
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Failure of eliminating apoptotic cells, due to a clearance defect or an overload of apoptotic
cells, allows the cells to proceed into secondary necrosis. This results in cell swelling and
eventually bursting, leading to uncontrolled leakage of noxious contents and autoantigens
(89). The induction of massive apoptosis in mice using anti-Fas antibodies was associated
with a severe inflammatory response (90). It has therefore been speculated that defective or
delayed clearance of apoptotic cells can result in the development of autoimmune diseases.
Systemic lupus erythematosus (SLE) is an autoimmune disease which is characterized by the
presence of autoantibodies against nuclear antigens such as DNA and RNA. Cystic fibrosis is
another disease, where an increased number of apoptotic cells in the airways is thought to be
the result of defective apoptotic cell clearance. This disease is characterized by inflammation
that is associated with a massive influx of inflammatory cells and the release of intracellular
proteases in the lung (90).
Senescent erythrocytes Erythrocytes originate from the bone marrow. A special feature of the erythrocytes is that they
lose their nucleus before they leave the bone marrow and enter circulation. Moreover, they
lack organelles such as endoplasmatic reticulum and mitochondria. Erythrocytes have a
biconcave shape and their main function is to deliver O2 to tissues, and to take up CO2 from
tissues and transport it to the lungs. They are highly viscous and can easily change shape
enabling them to squeeze through thin capillaries. Human erythrocytes have a lifespan of
about 120 days , before they are cleared from the circulation by macrophages (91).
Senescent murine erythrocytes are cleared from the circulation preferentially in the spleen
(92). These aging erythrocytes display some similar features with apoptotic cells, such as
vesiculation, cell shrinkage and PS exposure (93). However, since erythrocytes have no nuclei
or mitrochondria, the term eryptosis instead of apoptosis was suggested to describe their
senescence (94). Osmotic and oxidative stresses are known apoptotic triggers for both
nucleated cells and erythrocytes. Erythrocyte senescence can be mimicked in vitro by
treatment with a Ca2+ionophore, such as ionomycin, where an increase in intracellular the
Ca2+ concentration accelerates eryptosis (95)
As for apoptotic nucleated cells, recognition and clearance of aged erythrocytes is associated
with membrane alterations. Band 3 is quantitatively the major protein on the surface of
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erythrocytes and it is involved in the exchange of chloride and bicarbonate. In aging
erythrocytes, changes to band 3 enable binding of natural IgG antibodies and subsequent
phagocytosis. Although it is not entirely clear how band 3 changes during senescence, it may
implicate aggregation of band 3, or a partial breakdown of band 3 exposing the epitope for
IgG (93).
Other membrane alterations on aged erythrocytes include upregulated cell surface expression
of PS. As with apoptotic cells in general, this results in prophagocytic signalling upon contact
with phagocytic cells (96). Loss of membrane proteins involved in phagocytic inhibition is
another way to facilitate phagocytosis of senescent erythrocytes. Both siliac acid (97,98) and
CD47 (3) have been proposed to be markers of self on erythrocytes, since they inhibit
phagocytosis upon interaction with macrophage Siglecs and SIRPα, respectively.
Observations show loss of both siliac acid (99) and CD47 (100) on aged erythrocytes.
Moreover, CD47 may also to some extent be lost on erythrocytes stored for transfusion
(101,102).
Macrophages Monocytes are released from the bone marrow into the peripheral blood where they circulate
for several days before they enter different tissues and differentiate into specific tissue-
resident macrophage populations. Macrophages are located throughout the body and are
found in lymphoid organs, as well as in non-lymphoid organs such as liver (Kupffer cells),
lung (alveolar macrophages), nervous system (microglia), reproductive organs, and serosal
cavities. Moreover, macrophages can be found within the lamina propria of the gut and in the
interstitium of organs such as the heart, pancreas and kidney (103). In addition, monocytes
can give rise to specialised cells such as DCs and osteoclasts (103). Originally, the monocyte
was the only source from where macrophages were thought to originate. However, later
studies revealed that many tissue-resident macrophage populations, such as splenic white pulp
and marginal metallophilic macrophages, alveolar macrophages and liver Kupffer cells are
maintained through local proliferation. This is mainly the case under steady-state conditions,
whereas in inflammatory conditions recruitment of circulating monocytes is the major
contribution to the macrophage population at the specific location (104). Macrophages are
professional phagocytes that are involved in both innate and adaptive immune responses
20
(105,106). Apart from their role in pathogen clearance and the function as an antigen
presenting cell, macrophages are also important in the clearance of apoptotic cells (103).
Macrophage subpopulations
There is a high heterogeneity among both monocytes and macrophages concerning cell
surface receptor expression, localization, and function. The specialization of tissue
macrophages is a result of the particular microenvironment they end up in (106), hence the
microenvironment also has a role in deciding macrophage function (107). Heterogeneity can
be found both between different organ-specific macrophages, but also within a single organ
such as the spleen. In the spleen, there is a discrete anatomical localization of the different
macrophage populations. The marginal-zone macrophages, located in the splenic marginal
zone adjacent to the marginal sinus where the circulation passes through, express receptors
enabling them to aid in the clearance of blood borne pathogens. Marginal metallophilic
macrophages, located at the border between the white pulp and the marginal sinus, can sample
the circulation for viral infections and other infections. Tingible-body macrophages are
located in the splenic white pulp and are involved in the clearance of apoptotic cells (106),
and splenic red pulp macrophages are mainly implicated in the clearance of the majority of
senescent erythrocytes (92).
Dentritic cells DCs are professional antigen presenting cells with the ability to activate T cells and thereby
exert immune-regulatory functions. Immature DCs are present in most tissues and have a high
endocytic capacity to capture a number of different antigens, such as peptides, bacteria,
viruses, and dying cells. After encountering any of these antigens, the DCs undergo
maturation, which includes changes in the chemokine receptor repertoire enabling entry into
lymphatics and guidance into T cell areas in lymphoid tissues, redistribution of MCH class II
molecules to the surface, and upregulation of costimulatory molecules (e.g. CD40, CD80, and
CD86). In their mature state, DCs activate T cells and thereby stimulate T cell immunity
(108). Similar to macrophages, DCs also have the ability to eliminate apoptotic cells with
beneficial immunological effects, where DC uptake is important for peripheral tolerance
(108). Central tolerance is achieved in the thymus where autoreactive T cells are eliminated.
However, all self-antigens are not represented in the thymus since many proteins don’t have
access to the thymus during development (e.g. growth of new tissues such as breast during
21
puberty), making it possible that some self-reactive T cells escape selection in the thymus.
Peripheral tolerance is therefore of great importance to avoid activation and proliferation of
self-reactive T cells and subsequent autoimmune disorders (108). DCs phagocytose apoptotic
cells, degrade them, and cross present the digested self peptides from apoptotic cells on MHC
class I on their surface. By migrating to secondary lymphoid organs and presenting the
engulfed self-antigens to T cells, self-reactive T cells will become deleted or anergic to
achieve peripheral tolerance (86).
As for macrophages, there are a number of different subsets of DCs. In the spleen, three major
DC subpopulations can be identified based on the expression of CD8α, CD4, and CD11b: 1)
CD8α− CD4+ CD11b+, 2) CD8α+ CD4− CD11b-, and 3) CD8α− CD4− CD11b+ (109). It is the
CD8α+ CD4− CD11b- DCs that have been found to be important for the elimination of blood-
borne apoptotic cells. So far, two subpopulations of CD8α+ DCs have been identified in the
murine spleen, CD103+ DEC-205 (CD205)+ CD207 (langerin)+ DCs and CD103- CD205-
CD207- DCs. It is the CD103+ CD205+ CD207+ DC subpopulation, mainly present in the MZ,
that is responsible for elimination of blood-borne apoptotic cells (110). Depletion of marginal
zone macrophages resulted in a reduced uptake of apoptotic cells and an impaired tolerance to
cell-associated antigens and it was noted that macrophages in the marginal zone appeared to
regulate engulfment of apoptotic cells by CD8+ DCs (111). Moreover CD47 appears to
regulate trafficking and function of specific DC subsets, since CD47-deficient mice have an
almost total loss of the CD8- CD4+ CD11b+ DCs in the marginal zone (112).
Both non-professional and professional phagocytes can eliminate apoptotic cells. Immature
monocyte-derived DCs (iMoDC), granulocyte-macrophage colony-stimulating factor (GM-
CSF)-driven macrophages (MØ1s), and IL-10-producing M-CSF-driven macrophages
(MØ2s), are three professional phagocytes derived from the same monocyte population.
Comparison of their capacity to take up apoptotic cells revealed that the anti-inflammatory
MØ2 macrophages take up early apoptotic cells more efficiently than late apoptotic or
necrotic cells, and superior to the uptake by MØ1 and iMoDCs (113). It was suggested, that
under steady-state conditions, uptake of apoptotic cells is achieved by this specialized anti-
inflammatory MØ2 subset of phagocytes. In a situation of apoptotic overload, other subsets of
phagocytes, such as the pro-inflammatory MØ1, might act as backups. In the latter scenario,
22
appropriate opsonization may determine the consequence of the uptake, since most opsonins
bind to late apoptotic cells (107).
23
AIMS To study if phagocytosis could be stimulated by the trans interaction between LRP1
and CRT, and if the CD47/SIRPα-interaction could regulate this function.
To investigate if glucocorticoids could affect the expression level of LRP1 in
macrophages.
To investigate possible changes to CD47 on apoptotic cells.
To study the role of CD47 on experimentally senescent erythrocytes in regulating their
uptake both in vitro and in vivo.
24
RESULTS
LRP1 - a prophagocytic receptor Previous studies have shown that a cis interaction between LRP1 and CRT, where both LRP1
and CRT are present on the macrophage, stimulate uptake of apoptotic cells by the interaction
with the collagenous tails of collectin-family members (e.g. C1q and MBP) (31,45). In
addition CRT is found at various levels on the surface of most cells, raising the possibility of
a trans activation between LRP1 and CRT.
CRT on the apoptotic cells is involved in their uptake
To elucidate a possible role for CRT on the apoptotic cell in the elimination process, apoptotic
Jurkat T cells, human neutrophils, and mouse embryonic fibroblasts (MEFs), were treated
with a blocking antibody against CRT, which resulted in a reduced uptake by the non-
professional phagocyte MEFs (Fig.1A, paper I). A further approach to determine the role of
CRT in promoting uptake of apoptotic cells was to study uptake of apoptotic MEFs from
CRT-deficient mice. Again, phagocytosis of these targets by MEFs or by J774 macrophages
was reduced (Fig.1B, paper I). Direct addition of exogenous CRT to these CRT-deficient
apoptotic cells restored their CRT surface levels to that seen in apoptotic wild-type cells and
also restored their ability to be phagocytosed (Fig.1D, E, paper I). Another proof that CRT
could act as a direct stimulator of endocytosis was provided by the finding that exogenous
addition of CRT stimulated macropinocytosis in phagocytes (Fig.2A, paper I), and the
activation of Rac-1, a known intracellular signalling molecule mediating apoptotic cell uptake
(Fig.S5, paper I). Investigation of the role of CRT in vivo included examination of 13.5 day
embryos from CRT-deficient mice, or intraperitoneal injection of CRT-deficient apoptotic
MEFs and subsequent collection of peritoneal macrophages. Both approaches showed reduced
uptake of apoptotic cells in the absence of CRT (Fig.1G, H, paper I). These results
demonstrate that CRT on the apoptotic cells has a direct effect on their elimination by both
professional and non-professional phagocytes.
LRP1 is the CRT-receptor mediating phagocytosis
To investigate if LRP1 could interact with CRT on the apoptotic cells, and subsequently be
the receptor responsible for the observed CRT-stimulated cell uptake, binding of exogenous
CRT to LRP+/+ or LRP-/- MEFs was analyzed by flow cytometry. We found a reduced amount
of CRT on LRP-/- MEFs at baseline, but more importantly no further increase in surface CRT-
25
levels after addition of exogenous CRT (Fig.2F paper I). Moreover, a functional blocking
antibody against LRP1 (Fig.2E paper I), as well as RAP, which blocks the binding of several
LRP1-ligands and has been extensively used as an antagonist in the study of LRP1 (Fig.2G,
paper I), both reduced apoptotic cell uptake.
The expression level of LRP1 can be modulated by glucocorticoids and modulate
macrophage phagocytic capacity
Glucocorticoids (GCs) are used in the treatment of many inflammatory conditions. Apart from
observations where GCs inhibit recruitment of inflammatory cells, pro-inflammatory cytokine
production, or inflammatory cell responsiveness (114), GCs are found to enhance macrophage
phagocytosis of apoptotic cells (115,116). Since LRP1 appears to have a positive effect on
apoptotic cell uptake, we studied if the GC dexamethasone could affect macrophage LRP1
expression. Flow cytometric analysis revealed a time- and dose-dependent increase of LRP1
expression on peritoneal macrophages in response to dexamethasone (Fig.2B, C, paper II). In
addition to an overall increased LRP1 expression level on the dexamethasone-treated
macrophages, the population of macrophages expressing higher amounts of LRP1 (LRP1hi)
was also increased (Fig.2A, paper II). Moreover, it has been suggested that for GCs to
optimally stimulate apoptotic cell uptake by macrophages, GCs have to be added early on
during macrophage differentiation (115). Addition of dexamethasone to freshly isolated bone
marrow precursors during culture of bone marrow-derived macrophages (BMM) (day 0), or
after three days of culture (day 3), followed by flow cytometric analysis of LRP1-expression
after 6 days, showed that day 0 BMM had a higher LRP1 expression than day 3 BMM
(Fig.3B, paper II). Incubation with the GC-receptor antagonist RU486 abolished the
dexamethasone-induced LRP1 upregulation (Fig.4A, paper II). Dexamethasone-treated
resident peritoneal macrophages expressed the same total amount of LRP1 as untreated
macrophages (data not shown, paper II). Since we found that uptake of viable unopsonized
CD47-/-RBCs by resident peritoneal macrophages is to a large extent mediated by LRP1
(Fig.5A, B, C, paper I), we incubated CD47-/-RBCs with untreated or dexamethasone-treated
resident peritoneal macrophages to investigate if dexamethasone-induced LRP1-upregulation
increased the phagocytosis of CD47-/-RBCs. These experiments indeed showed that
dexamethasone-treated macrophages were more efficient in their uptake of CD47-/-RBCs, as
compared with untreated macrophages (Fig.6, paper II), indicating a possible role for the
upregualted LRP1 expression in the observed increase of apoptotic cell uptake following GC
treatment.
26
Changed distribution and expression of CD47 and CRT on
apoptotic cells Modifications of protein expression patterns on the surface of apoptotic cells are important for
their efficient removal by phagocytes. Since the trans-interaction between LRP1 and CRT
was found to induce “eat me” signals, the CD47/SIRPα interaction mediates “don’t eat me”
signals, and the fact that both CRT and CD47 are present on viable cells, we speculated that
there might be changes to these proteins on apoptotic cells to facilitate their uptake.
CRT expression is upregulated and redistributed on apoptotic cells
Flow cytometric analysis of a number of cell types (e.g. fibroblasts and Jurkat T cells) showed
an increased expression of CRT after apoptotic induction with different stimuli (Fig.3A, paper
I). Neutrophils also upregulated their surface expression of CRT and showed a possible
relationship between expression levels of CRT and degree of apoptosis (Fig.3B, paper I). In
contrast to the fairly even distribution of CRT on the surface of viable cells, CRT on apoptotic
cells appeared to be redistributed into patches on the cell surface (Fig.3C, paper I)
Changes to CD47 on the surface of apoptotic cells differ between cell types
The surface levels of CD47 was found to be downregulated on apoptotic fibroblasts (Fig.6A,
paper I) and neutrophils (Fig.6B, paper I), but not on apoptotic Jurkat T cells (Fig.6C, paper
I). As for CRT, CD47 was also redistributed into patches on apoptotic fibroblasts, neutrophils
and Jurkat T cells (Fig.6D, paper I).
CD47 on apoptotic murine thymocytes facilitates their uptake by mediating binding of
apoptotic cells to the macrophages
As mentioned above, it is well established that CD47 on viable cells protects them from
phagocytosis. Interestingly, and contradictory, we found a reduced uptake of apoptotic murine
CD47-/- thymocytes by resident peritoneal macrophages, both in vitro (Fig.2B, paper III) and
in vivo (Fig.2C, paper III), and by BMM in vitro (Fig.2E, paper III), as compared to that for
equally apoptotic wild-type thymocytes. This difference in uptake of apoptotic wild-type and
CD47-/- thymocytes was observed during PS-dependent uptake (Fig.2A paper III) as well as
during PS-independent uptake (Fig.2D, paper III). However, the positive effect of CD47 on
phagocytosis of apoptotic cells was only observed using non-activated BMM, but not in β-
1,3-glucan-activated BMM (Fig.2F paper III). Binding of the apoptotic cell to the phagocyte
27
is one of the initial and important steps in the phagocytosis process (27). By using BMM and
apoptotic wild-type or CD47-/- thymocytes, we could detect a significantly lower binding of
CD47-/- thymocytes to the macrophages, as compared with wild-type thymocytes (Fig.3A,
paper III). This function of CD47 to enhance binding of apoptotic cells to the macrophages
did not require F-actin polymerization in the macrophage (Fig.3, paper III), since it was
insensitive to the F-actin-disrupting drug cytochalasin D.
Redistribution of CRT and CD47 into certain areas on the apoptotic cell surface
As mentioned above, both CRT and CD47 are redistributed on the surface of apoptotic cells.
Redistribution and concentration of proteins into specific plasma membrane domains could
serve two purposes: 1) to affect their interaction with other proteins (e.g. receptors on the
macrophage), or 2) to segregate individual groups of proteins away from each other within the
plasma membrane. On the surface of apoptotic Jurkat T cells, CD47 was found to segregate
away from CRT (Fig.6E, paper I). CRT co-localized with PS in cholesterol rich, GM-1+
ganglioside-containing rafts on apoptotic neutrophils (Fig.7C paper I). In this way,
prophagocytic ligands were clustered in certain areas of the plasma membrane, while CD47
was accumulating in other domains of the plasma membrane (Fig.6E, S12, paper I). However
in paper III, where apoptotic murine thymocytes were investigated, CD47 was found in cell
surface clusters which were both separated from and colocalized with PS. Patching of CD47
on apoptotic cells did not alter its ability to bind SIRPα (data not shown, paper I). However, in
contrast to that seen following interaction between macrophages and viable cells, western blot
analysis showed that macrophage SIRPα was not tyrosine phosphorylated upon contact with
apoptotic cells (Fig.6F, paper I).
Role of CD47 in the phagocytosis of experimentally senescent
erythrocytes Senescent erythrocytes display various features similar to apoptotic nucleated cells, such as
PS exposure and cell shrinkage (91,93,94). The Ca2+-ionophore ionomycin can be used to
induce experimentally senescent PS+ erythrocytes (Ca2+-RBCs) (94). We used this approach
to study the mechanisms behind phagocytosis of experimentally senescent erythrocytes in
vitro and in vivo, and the role of CD47 in this process.
28
CD47 does not affect phagocytosis of ionomycin-treated PS+ Ca2+-RBCs in vitro
Following treatment with ionomycin, wild-type or CD47-/- erythrocytes were found to expose
PS to a similar extent (Fig.1B, paper IV). In contrast to the enhanced phagocytosis of IgG or
complement-opsonized CD47-/- erythrocytes, as compared with equally opsonized wild-type
erythrocytes (64,75), we found no difference in macrophage phagocytosis of wild-type or
CD47-/- Ca2+-RBCs by resident peritoneal macrophages in vitro (Fig.1C, paper IV). A
previous study showed that phagocytosis of oxidatively damaged erythrocytes is in part
sensitive to inhibition of ERK1/2, PI3 kinase, or Syk tyrosine kinase, but insensitive to
inhibition of Src-family kinases (117). However, the mechanism behind uptake of Ca2+-RBCs
did not involve ERK1/2, Syk, or Src-family kinases, since no significant phagocytosis
inhibition was found when using specific inhibitors. In contrast, a complete inhibition of
phagocytosis was detected upon using the PI3 kinase inihibitor LY294002 (Fig.2B, C, paper
IV).
In vivo clearance of experimentally senescent erythrocytes
The mechanisms behind the in vivo clearance of experimentally senescent Ca2+-RBCs in vivo
are poorly understood (118). Therefore, we injected wild-type mice with wild-type PKH26-
labeled Ca2+-RBCs and studied spleen sections by immunohistochemistry after 1 hour and 20
hours. Already 1 hour after injection, a considerable amount of the injected Ca2+-RBCs were
found in the marginal zone (Fig.3A, paper IV) ), co-localized with MARCO+ marginal zone
macrophages (Fig.3C, paper IV), and to some extent with MOMA-1+ marginal metallophilic
macrophages (Fig.3D, paper IV). Besides macrophage uptake of these Ca2+-RBCs, a small
amount was also phagocytosed by DCs in the marginal zone and in the bridging channels
(Fig.3E, paper IV). By comparing uptake after 1 hour and 20 hours we revealed some red
fluorescence from the injected RBCs in the T cell area of the splenic white pulp at the later
time-point, which was not found at 1 hour (Fig.4A, B, paper IV). In addition to marginal zone
macrophages, DCs are also known to be involved in the elimination of apoptotic cells in the
spleen and have an important role in the peripheral self-tolerance (86,87,108). Therefore, we
next investigated the role of DCs in uptake of Ca2+-RBCs at 20 hours after intravenous
injection of PKH26 labeled wild-type Ca2+RBCs into wild-type recipients, using
immunohistochemical labeling of CD11c on spleen sections (Fig.4C, paper IV) and flow
cytometric analysis of CD11chi cells from collagenase-digested spleens (Fig.5A, paper IV).
The immunohistochemical analysis showed co-localization of PKH26-fluorescence and
CD11c staining in both the marginal zone and in the T cell area (Fig.4C, paper IV). Flow
29
cytometric analysis indicated that 20-25% of the CD11chi cells were positive for PKH26
(Fig.5A, paper IV). Interestingly, the spleen contains several DC subsets, among which CD8+
DCs constitutes about 20% of all splenic DCs (Fig.5B, paper IV; (109,112)). This subset has
also been shown to mediate uptake of apoptotic T cells (110,119). By gating on CD8+
CD11chi cells, we found that around 80% of the cells in this subset were strongly positive for
PKH26 (Fig.5B, paper IV). Further analysis of spleens by immunohistochemistry showed that
scattered CD207+ cells in the marginal zone were positive for uptake of Ca2+-RBCs at 1 hour
and 20 hours after injection, and that CD207+ cells in the T cell areas were associated with
injected Ca2+-RBCs at the 20 hour time-point (Fig.6, paper IV). DC expression levels of MHC
class II and co-stimulatory molecules is important for their interaction with T cells (120).
Therefore, the expression levels of MHC class II, CD40 and CD86 was investigated and
compared in splenic CD11chi DCs which were positive or negative for uptake of PKH26
labeled wild-type Ca2+RBCs. This analysis revealed a slightly increased expression of MHC
class II, CD40 and CD86 in PKH26+ DCs, as compared with DCs negative for uptake of
Ca2+-RBCs (Fig.7, paper IV). When comparing DC uptake of wild-type and CD47-/- Ca2+-
RBCs in vivo, we found similar uptake of both genotypes in wild-type recipients (Fig.8, paper
IV). Wild-type splenic DCs which had taken up wild-type or CD47-/- Ca2+-RBCs showed
identical expression levels of MHC class II, CD40 and CD86. However, an unexpected
finding in wild-type recipients of CD47-/- Ca2+-RBCs was a marked increase in CD86
expression in DCs negative for uptake of CD47-/- Ca2+-RBCs (Fig.9, paper IV).
30
DISCUSSION Identification of cell surface proteins involved in phagocytosis and removal of apoptotic cells
has been found to be challenging. There appears to be a redundancy in the system, since the
inhibition of any single known cell surface ligand on apoptotic cells can not completely
abolish phagocytosis of apoptotic cells. Apoptotic cell elimination is a process involving up
and down regulation of molecules on the apoptotic cell surface, as well as their redistribution.
The intention of this seems to be the modulation of phagocytosis through activating or
inhibitory receptors, to make sure that the activating signal is greater than the inhibitory
signal. The work presented in this thesis will hopefully help to further understand the
mechanisms involved in regulating these processes during uptake of apoptotic cells.
LRP1/CRT interaction and apoptotic cell removal CRT was found to be upregulated and redistributed on the cell surface during development of
apoptosis, and able to induce uptake of apoptotic cells. LRP1 was found to be the phagocyte
receptor for CRT and responsible for the positive effect on apoptotic cell removal. For surface
expression, CRT has to associate with surface structures since it lacks a transmembrane
domain (121). Depending on the structure CRT associates with, different outcomes may be
possible. Little is known about the structures that mediate binding of CRT to the surface of
viable or apoptotic cells. However, CRT has been shown to bind to the GPI-anchored
complement regulatory protein CD59 on viable human neutrophils (121). Although the
expression levels of CD59 may vary among different subsets of blood cells (122), our
preliminary data shows that erythrocytes express high levels of CRT. The fact that
erythrocytes also express high levels of CD59 makes it intriguing to speculate that CD59
mediates a significant part of the binding of CRT to the surface of blood cells. Our
experiments where exogenous CRT was added to the cells indicated a limited availability of
free binding sites for CRT on the surface of apoptotic cells, since the amount of CRT on the
surface of apoptotic cells appeared to be saturated (Fig.1D, paper I). CRT was found to be
redistributed into patches on apoptotic cells. The addition of CRT to apoptotic CRT-/- cells
also resulted in a patched distribution, indicating that the binding partner may redistribute as
well. One aspect of the observed increase in the number of apoptotic cells in CRT-/- embryos,
both in our study and that by Rauch et al. (123), is that we cannot be sure that this is indeed
due to an impaired phagocytosis and not because of a higher rate of apoptosis. However, in
vitro culture of CRT-/- MEFs did not show an increased rate of apoptosis (123), strengthening
31
the hypothesis that lack of CRT primarily results in impaired removal of apoptotic cells.
Although several cell surface changes has been implicated in apoptotic cell recognition
(124,125), upregulated cell surface PS is still the single ligand with a wide distribution
between cell types and across animal phyla. Similarly, CRT is a highly conserved protein,
present on the surface of most mammalian cells (126-128). Therefore, our finding that CRT
expression on apoptotic cells mediates interaction with the conserved receptor LRP1 to induce
apoptotic cell removal, makes CRT a possible candidate of an additional “general” ligand
such as PS. Given the number of biological functions involving CRT and LRP1, and the fact
that they are both highly conserved, it is not surprising that their deletion in mice is lethal.
Altered protein expression levels on the apoptotic cell surface affect the response of
phagocytes upon contact. Likewise, alterations of phagocyte expression levels of receptors
affect their responses. As an example, macrophage SIRPα expression has been found to be
downregulated by lipopolysaccharide (LPS), which would possibly facilitate an innate
immune response (74). On the other hand, SIRPα was found to be upregulated on GC treated
macrophages, which was associated with inhibition of their proliferation (129). Among other
things, one effect of anti-inflammatory GCs is an increased phagocytosis of apoptotic cells
(114-116). Our results indicate that this could be due to an increased expression level of the
phagocyte receptor LRP1. Flow cytometric analyses revealed a small population of
macrophages expressing higher amounts of LRP1. In addition to the overall increase in LRP1,
the macrophage population expressing high levels of LRP1 increased upon treatment with the
GC dexamethasone. Interestingly, in phagocytosis experiments with dexamethasone-treated
and non-treated macrophages, the fraction of macrophages ingesting apoptotic cells was
comparable to the fraction of LRPhi macrophages in each condition. These data indirectly
suggest that the LRP1hi macrophages are the macrophages responsible for the apoptotic cell
uptake. The mechanism behind the observed LRP1 upregulation could be carried out by either
the classical genomic mechanism, involving the passage of dexamethasone through the
plasma membrane, interaction with cGCR, and transportation to the nucleus where either
transactivation or transrepression of specific genes occur, or via the non-genomic
mechanisms. There are three suggested non-genomic mechanisms that are explored in an
attempt to explain the rapid effects of GC that cannot be due to the genomic mechanism
(130). A significant upregulation of LRP1 in response to dexamethasone was not detected
until about 12h of incubation. This opens up the possibility of a genomic mechanism.
However, even though we found a slight increase in LRP1 mRNA (unpublished data), the
32
total amounts of LRP1 at the protein level were the same for dexamethasone-treated as for
non-treated macrophages. Therefore the observed upregulation of cell surface LRP1 appears
to primarily be the result of transportation of pre existing LRP1 to the plasma membrane and
consequently more likely the result of a non-genomic mechanism. Since our findings show
that LRP1 can mediate macrophage uptake of viable unopsonized CD47-/- erythrocytes (paper
I), the evidence for a direct role of elevated LRP1 expression on the increased phagocytosis of
apoptotic cells is supported by the enhanced uptake of CD47-/- erythrocytes by
dexamethasone-treated macrophages. Furthermore, the reported upregulation of SIRPα
following GC treatment (129) could be an additional factor facilitating increased uptake of
apoptotic cells, since the CD47/SIRPα-interaction appears to promote binding and uptake of
apoptotic cells ((131); paper III).
CD47 on apoptotic cells The fact that CRT is also present on viable cells, suggests regulatory mechanisms to avoid
elimination of viable cells. The upregulation and redistribution of CRT on apoptotic cells may
enhance its ability to stimulate LRP1. However, this is not likely to be the only explanation
behind the fact that viable cells are not eliminated despite their sometimes high CRT
expression. Interaction between SIRPα and CD47 on viable cells inhibits phagocytosis (3).
CD47 is therefore thought of as a “marker of self” that inhibits uptake of viable cells. It would
be expected that CD47 is altered on apoptotic cells to decrease the inhibitory signal and
enable uptake. Indeed we found a downregulated expression of CD47 on apoptotic fibroblasts
and neutrophils, but not on apoptotic Jurkat T cells or apoptotic murine thymocytes. In
addition, and similar to CRT, CD47 was redistributed into patches on the apoptotic cell
surface. An expected effect of total loss of CD47 would then be increased uptake due to the
loss of the CD47/SIRPα-induced phagocytosis inhibition. However apoptotic murine CD47-/-
thymocytes were found to be phagocytosed to a lower extent by non-activated macrophages,
as compared with equally apoptotic wild-type thymocytes. Uptake mechanisms are different
depending on the macrophage phenotype (132), and we found that uptake by activated-
macrophages was not affected by CD47 on the apoptotic cell. These findings are supported by
results from Tada et al (131), where the BAM3 macrophage cell-line, but not thioglycolate-
activated peritoneal macrophages, showed impaired phagocytosis of apoptotic WR19L
lymphoma cells (deficient in CD47 expression), as compared with apoptotic wild-type
thymocytes. As a result, Tada et al. suggested that CD47 may not at all function as a “marker
33
— isotype control— viable Jurkat T cells— apoptotic Jurkat T cells
SIRPα-Fc binding
Figure 4. Binding of SIRPα Fc-fusion protein to viable or apoptotic Jurkat T cells. Cells were incubated with SIRPα-Fc, followed by a PE-conjugated anti-human Fc-specific secondary antibody, and analysis by flow cytometry.
of self” on lymphocytes (131). However,
our results clearly show that CD47 on
viable IgG-opsonized thymocytes can
potently inhibit macrophage
phagocytosis (paper III). In addition we
found that CD47 on apoptotic
thymocytes promoted both PS-
dependent and PS-independent uptake,
and a binding assay showed decreased
binding of apoptotic CD47-/- thymocytes
to non-activated macrophages.
The question is how CD47 goes from being a marker of self, saving viable cells from
elimination, to promoting uptake of apoptotic cells. The answer might be in the observed
redistribution of CD47 into patches on the apoptotic cell surface, which could possible alter
the availability of the binding site for SIRPα. However, although CD47 on apoptotic cells
show no defect in the ability of binding to SIRPα (131) (Fig.4-unpublished data), no
phosphorylation of the receptor was detected upon contact between the receptors (paper I).
Besides the known recruitment of the SHP-1 and SHP-2 phosphatases upon CD47 interaction
with SIRPα, downstream targets and mechanisms in phagocytosis inhibition has been unclear.
The process of FcγR-mediated phagocytosis involves the adhesion, pseudopod extension, and
internalization of the phagocytic prey into a phagosome. This requires cytoskeletal assembly
around the particle to be internalized, depending on accumulation of F-actin among other
components (133). Non-muscle myosins and their phosphorylation, play a role as contractile
motors during internalization (134). Since it was shown that engagement of SIRPα by CD47
results in a decreased myosin II phosphorylation (63), myosin II was put forward as a possible
target for the phagocytosis inhibition observed upon contact between macrophages and viable
cells. Although the mechanisms behind internalization of apoptotic cells might differ from
FcγR-induced internalization, inability of CD47 on apoptotic cells to induce SIRPα
phosphorylation would then theoretically allow myosin II phosphorylation and subsequent
internalization. In addition to this, the physical binding of CD47 to SIRPα on the phagocyte
further promotes uptake. It is not known why phosphoylation of SIRPα does not occur upon
contact with CD47 on apoptotic cells. CD47 may possibly interact with another ligand than
SIRPα on the macrophages. However, antibody-blocking of SIRPα, or the presence of soluble
34
SIRPα, but not SIRPα constructs lacking the IgV-domain, necessary for CD47 binding,
inhibited phagocytosis of apoptotic cells (131). Thus, it is likely that CD47 on the apoptotic
cells indeed interacts with SIRPα on the macrophage to enhance uptake.
Lipid rafts are platforms present in the plasma membrane, containing high amounts of
cholersterol and different lipids, as well as proteins. These rafts can fuse together and form
large platforms (135). We observed a co-localization of PS and CRT an apototic cells and
they were found in GM-1 ganglioside-containing rafts. However CD47 was not detected in
these GM-1 lipid rafts together with PS and CRT (paper I). This was observed in apoptotic
human neutrophils and Jurkat T cells. Further studies (paper III) showed that CD47 on
apoptotic cells may co-localize with PS and GM-1 domains. Since the latter observation was
made in murine thymocytes, the different results might be explained by a species difference.
The clustering of CD47 might be a result of lipid raft fusion which is supported by the
reduced clustering of CD47 and PS following cholersterol depletion with cycklodextrin
(unpublished observation). Even though CD47 was not colocalized with CRT and PS in GM-
1 domains on the human apoptotic cells, CD47 might still be present in lipid rafts containing
other lipids that fuse to form CD47 patches.
Clearance of experimentally senescent erythrocytes We found that CD47 appears to have no impact on the phagocytosis of experimentally
senescent PS+ erythrocytes (Ca2+-RBCs). This was observed both by macrophages in vitro
and by DCs in vivo.
Experimentally senescent erythrocytes have some similarities with apoptotic nucleated cells,
for instance PS exposure and cell shrinkage (93). Since injected apoptotic T cells have been
found to be eliminated by macrophages and DCs in the marginal zone of the spleen
(110,111,136) and that this uptake was important for self-tolerance (137), it was interesting to
note that the same phagocytes were responsible for uptake of Ca2+-RBCs. This opens up the
possibility that uptake of Ca2+-RBCs may also be important for induction of self-tolerance to
erythrocyte antigens. The general idea is that aged erythrocytes are eliminated by
macrophages in the splenic red pulp (92). The fact that our experimentally senescent
erythrocytes were mainly taken up by MARCO+ marginal zone macrophages and DCs in the
marginal zone, while very few were detected in the red pulp, could be due to the expression of
35
age-associated cellular changes, in addition to PS, on physiologically senescent erythrocytes
(91). It might be these age-associated cellular changes that result in a preferential uptake by
red pulp macrophages. However, PS exposure is considered a central event in the regulation
of uptake of most cells and studies on Ca2+-RBC uptake could therefore still be valuable for
evaluating the ability of erythrocytes to modulate the immune system.
CD8α+ DCs have traditionally been considered to reside mainly in the T cell area of the
splenic white pulp. However, recently a subset of CD8α+ DEC-205 (CD205)+ CD207
(langerin)+ DCs was discovered in the marginal zone of the spleen (138). In the marginal
zone, two subsets of phagocytes have been shown to be important for the uptake of blood-
borne apoptotic cells, namely the marginal zone macrophages and the CD8α+ CD103+
CD207+ DCs (110,111). We found that uptake of Ca2+-RBCs was carried out by the same
phagocyte populations. These phagocyte populations appears to be important in tolerance
induction, since elimination of either of these phagocyte populations abrogates tolerance
(110,111). Interestingly the process of sustaining peripheral tolerance might involve
regulatory T cells, since oral tolerance function of CD103+ DCs in the gut was found to be
mediated by the induction of regulatory Foxp3+ CD25+ T cells (139). Noteworthy for our
studies was that CD8α+ CD205+ DCs were also found to induce regulatory Foxp3+ T cells in
the spleen. To place the importance of regulatory T cells in an autoimmune disease context, it
was observed that an almost complete deletion of regulatory CD4+ CD25+ T cells markedly
enhanced the development of the autoimmune hemolytic anemia (AIHA), which further
support the role of regulatory T cells in tolerance induction (140). Therefore it would be of
interest to study if intravenously injected Ca2+-RBC could increase the formation of
regulatory T cells as part of inducing tolerance.
CD47 appeared to have no impact on the uptake of Ca2+-RBCs, neither by macrophages in
vitro nor by DCs in vivo. It is known that CD47 on viable cells function as a marker of self,
since phagocytosis of these cells by macrophages and DCs is inhibited (2). The fact that
CD47 did not inhibit DC uptake of Ca2+-RBCs in vivo may not be surprising, since splenic
CD8α+ DCs express marginal, if any SIRPα (112). However, the work presented in this thesis
(paper III) show that CD47 on apoptotic cells rather promotes phagocytosis in some
situations. In addition, CD47 has been found to have no effect on macrophage uptake of
oxidatively damaged RBCs (117). These results support our data that CD47 does not inhibit
phagocytosis of Ca2+-RBCs. Furthermore, CD47 may have another function in the elimination
36
of senescent RBCs. MHC class II and costimulatory molecules (e.g. CD40 and CD86) are
upregulated on DCs upon activation by microbal antigens (141). These markers were also
slightly upregulated on DCs positive for Ca2+-RBC uptake. Remarkably, in Wt mice injected
with CD47-/- Ca2+-RBCs, DCs that had not taken up any of these RBCs showed an increased
expression of the activation marker CD86, compared to DCs negative for uptake in recipients
of wild-type Ca2+-RBCs. Even though CD47 did not regulate uptake of Ca2+-RBC, the
expression of CD47 might therefore still modulate the activation status of DCs. This indirect
regulation of DC activation could well be mediated by another cell population in the marginal
zone, such as the marginal zone macrophages, since there is a described intimate connection
between this macrophage population and DCs in the process of apoptotic cell uptake and
tolerance induction (111).
37
CONCLUSIONS
CRT is an important recognition ligand on apoptotic cells that mediate engulfment by
activating the internalization receptor LRP1 on the phagocyte. This interaction
stimulates Rac-1 that drives the engulfment. Elimination of viable cells, found to
express CRT, was inhibited by the CD47/SIRPα interaction
LRP1 is upregulated on macrophages following treatment with the GC
dexamethasone. An increased LRP1 expression might partially explain the observed
elevated uptake of apoptotic cells following GC treatment. This conclusion is
supported by the increased uptake of CD47-/- RBCs by dexamethasone-treated
macrophages since uptake of these RBCs is dependent on LRP1 signaling.
CD47 on apoptotic cells appears to promote uptake instead of functioning as a marker
of self, as is the case on viable cells where CD47 inhibits uptake by interacting with
SIRPα. However, promoted uptake was only found with non-activated macrophages
and not activated macrophages, where apoptotic Wt and CD47-/- thymocytes were
taken up equally. The redistribution and clustering of CD47 into patches on apoptotic
cells could be of importance for CD47 to function more as tethering ligand, enhancing
the binding of the apoptotic cell to the phagocyte.
CD47 does not inhibit the uptake of experimentally senescent PS+ erythrocytess in
vitro or in vivo. PS+ erythrocytes are eliminated by splenic marginal zone
macrophages and CD11chi CD8α+ DCs, the same subset of phagocytes in the spleen
that also phagocytose apoptotic T cells and mediate tolerance induction.
In one day over 100-200 billion cells in the human body turns apoptotic and has to be
eliminated to avoid immunological consequences (63). There is still much work to be done to
understand the complex process of elimination of apoptotic cells and the observed induction
of tolerance in response to apoptotic cell uptake by DCs. The responses following apoptotic
cell uptake are regulated by the numerous interactions occurring between the phagocyte and
the apoptotic cells. Hopefully the results from this thesis have given some new clues to the
whole process.
38
ACKNOWLEDGEMENTS First of all I want to thank my supervisor Per-Arne Oldenborg for allowing me to be
part of his work team and for being such an inspiring and great teacher. For always believing in me and encouraging me when things were not going as planned. I could not have had a better supervisor.
Janove Sehlin, head of the department, for making me feel so welcomed and also for inviting me to participate at the conference in Archangelsk, Russia.
Thank you to all the people at IMB for making it such a nice place to work at and for always being available if I needed help. Special thanks to Gerd Larsson-Nyrén, Per Lindström, Gunilla Forsgren, Eva Boström, Solveig Persson-Sjögren, Eva-Maj Lindström, Eva Hallin, Karin Grabbe and Anita Dreyer-Perkiömäki.
Göran Dahlström, for computer support, especially at the end of this when my computer were working probably as slow as the first computer out on the market did.
Ann-Charlotte Idahl and Barbro Borgström, I am so grateful for the excellent technical assistance that you have helped me with over the years. You have been absolutely wonderful.
Doris Johansson, for all the help with handling the animals and assisting me when I was insecure of certain procedures. Thank you also to Mats Högström and Thomas Jonsson at the animal facility.
Ingrid Strömberg, for being so helpful, caring and encouraging, it has meant a lot to me.
Professor Peter M. Henson, at the National Jewish Medical and Research Center, Denver, CO, USA, for the very educational and pleasant weeks I was fortunate to spend in your lab. Also, thank you to the other collaborators on my first paper.
Thanks to all my lovely workmates at the 6th floor for making my years at IMB such a pleasant time with fruitful discussions and laughter. Sara what would I have done without you and your little notes and drawings. Thank you for trying to make me believe in myself and also for being the best dance partner, ever. Mattias, my personal technical and computer support, you have been a great roommate both on the 5th and 6th floor over the years. I will try to learn from your ability to never get nervous about things. Elisabeth, who I have known since I started my education here at Umeå University. I am glad that we ended up doing our PhDs at the same place. I enjoyed having you as a roommate with all our talks. Thanks for always listening and being happy. Franziska, my roommate for the last two years, thank you for always being so joyful and understanding, for introducing me to boxercise and for understanding the need for sweets Also, I owe you since you saved me from eating a “skylt”. Åsa, for all the nice talks about the small and big things in life. Nina, you were a fresh spirit coming into our lab, let’s get together some time and go crazy in Forever 21. Anders,
39
you were the help I so desperately needed at the end of this. Thank you for working so hard. I am glad you joined team PA. Anna R, you are such a kind person and I am happy I got to know you. Sven, for taking care of me when I came here, teaching me some of the techniques and making sure I understood the importance of mixing solutions correctly. Liselotte, my experienced researcher roommate for a couple of years, for all the discussions about training but also about research, sometimes. Ing-Marie, Anna B, Peter S, Martin, Heather and Mikael A, thank you for all the nice times both in the lab and outside the lab.
All my friends outside of the office, you all mean so much to me. Marie and Emma we have shared so much together and my stomach always hurts after seeing you from all the laughter. Martin, Malin, Samuel and Sara I don’t know if we will win the gingerbread house competition this year due to lack of time for preparation, so this year it is your time to shine. My dear friends with whom I have dinners with every other Wednesday, Kristina, Christina, Inger, Sofia, Linda and Carina (and also Magdalena and Linda L who join us if you are in town), thank you for being there for me and also for not saying anything about the fact that I haven’t had a Wednesday dinner at my place this fall. You will all be welcome to dinner the next Wednesday dinner after the 17th of December. I am afraid I can’t mention you all here, so thank you to all of my friends who make my life so rich.
I also want to thank all of my wonderful relatives with whom I have so much fun with, for being so helpful and caring.
A big thank you to Eva and Olle for always helping me and my family with whatever we need help with and for all the nice times out in the summerhouse and Bäck.
To my father Ulf and Agneta, for always encouraging me and believing in my abilities, that mean more to me than you know. Thank you also for calling me, when I forget, to make sure that everything is ok.
To my mother Inga-Maj and Knut, I have you to thank for so much and I would not be where I am right now without your support. Thank you for making me feel so loved.
My beautiful sisters Elin and Helena, thank you for taking my mind of research and focusing more on things like shopping and sweets. I absolutely love spending time with you, no matter what we are doing.
Stefan you are the best support one could ever ask for. Thank you for pushing me to aim higher and for loving me unconditionally. You mean the world to me. Hjalmar my little son, I love you so much, words cannot describe. Coming home, with you standing on top of the stairs, excited to see me or having you run towards me when I pick you up from the daycare centre, is the best feeling ever and all eventual worries about work always disappear. Jag älskar er så mycket!!!
Finally I would like to thank all of you who I unintentionally might have forgotten to mention.
40
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