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Mechanisms Involved in Macrophage Phagocytosis of Apoptotic Cells

Mechanisms Involved in Macrophage Phagocytosis of Apoptotic Cells

Mechanisms involved in of apoptotic cells

Anna Nilsson

Department of Integrative Medical Section for and Biology Umeå University, Umeå, Sweden 2009

Cover illustration: Confocal microscopic image of murine resident peritoneal , stained for F- (green), CMFDA labeled apoptotic (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 ...... 9 Elimination of apoptotic cells - an overview...... 10 “Find me signals”...... 11 “Eat me signals”...... 12 The /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 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 4

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 RBC red cells BMM marrow-derived macrophages RPM resident peritoneal macrophages GC GCR glucocorticoid receptor cGCR cytosolic glucocorticoid receptor Dex dexamethasone DC ITIM immunoreceptor tyrosine-based inhibitory motifs GM-CSF -macrophage colony-stimulating factor M-CSF macrophage colony-stimulating factor MEF mouse embryonic MZ marginal zone TSP thrombospondin Wt wild-type Ca2+-RBCs ionomycin-treated experimentally senescent PS+ erythrocytes AIHA autoimmune MHC major histocompatibility complex

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ABSTRACT Efficient removal of apoptotic cells is critical for development, tissue remodelling, maintenance of , 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 , can act as a receptor for 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 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. 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 ( and , 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 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 , 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, , 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 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

9 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 , 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 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 where degradation occurs (34). Degradation of apoptotic cells by DCs plays an important role in the maintenance of the (35).

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a) Soluble factors released from the b) Induction of eat me and don’t eat d) Engulfment apoptotic cell induces find me signals. me signals upon contact.

Apoptotic Apoptotic cell cell

Apoptotic PhagocytePhagocyte cell

Phagocyte

c) A majority of eat me signals activates phagocytosis

Don’t eat me Eat me

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 (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 -activating polypeptide ІІ (EMAP ІІ) is the product of another cleavage occurring during apoptosis and serves as a ligand for the -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), -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 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 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 presentation and complement activation, immunogenicity of death, wound , 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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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/ (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; , 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 for subsequent degradation. Ligands range from lipoproteins, proteins, protease/protease-inhibitor complexes, and , to and growth factors (54). The regulatory functions of LRP1 include controlling vascular smooth proliferation Figure 2. The CRT/LRP1 cell surface complex Cis interaction between CRT and LRP1on the (55) and regulating blood-brain barrier phagocyte function as a binding site for the collagenous tails of collectin-family members. C1q and SP-A are permeability (56). Moreover, LRP1 on members of the collectin-family that have the ability to bind to apoptoic cells and thereby facilitate their neurons regulate calcium signalling, an elimination. important second messenger for neuronal Reprinted by permission from Macmillan Publishers Ltd: Nat Rev Mol Cell Biol (1), copyright (2001) response to excitatory neurotransmitters

13 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 (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

14 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.

Table 1. Biological functions of CD47

Ligand/associated protein Function Reference SIRPα Marker of self, inhibits phagocytosis (3,4) SIRPα Regulaion of (5) SIRPα Macrophage fusion and formation (6) α-CD47 mAbs (9-13) SIRPα/γ Induction of apoptosis TSP-1 FAS/CD95-association

SIRPα, TSP-1 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)

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

15 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).

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.

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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 (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 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 (86,87). Interestingly, a study showed that transfusion of apoptotic 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 . 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 which is characterized by the presence of against nuclear antigens such as DNA and RNA. Cystic 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 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 . 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 . They are highly viscous and can easily change shape enabling them to squeeze through thin . 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 (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

18 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 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 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 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 (Kupffer cells), lung (alveolar macrophages), nervous system (), reproductive organs, and serosal cavities. Moreover, macrophages can be found within the propria of the gut and in the interstitium of organs such as the heart, pancreas and (103). In addition, monocytes can give rise to specialised cells such as DCs and (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 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

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(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 . Marginal metallophilic macrophages, located at the border between the and the marginal sinus, can sample the circulation for viral 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 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, , viruses, and dying cells. After encountering any of these antigens, the DCs undergo maturation, which includes changes in the 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 (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). is achieved in the 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

20 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 ()+ 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,

21 appropriate opsonization may determine the consequence of the uptake, since most bind to late apoptotic cells (107).

22

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.

23

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 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 . We found a reduced amount of CRT on LRP-/- MEFs at baseline, but more importantly no further increase in surface CRT-

24 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 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.

25

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

26 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 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.

27

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 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 -digested (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

28 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).

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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

30 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 (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

31 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

32 of self” on lymphocytes (131). However, — control — viable Jurkat T cells our results clearly show that CD47 on — apoptotic Jurkat T cells 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, SIRPα-Fc binding and a binding assay showed decreased Figure 4. Binding of SIRPα Fc-fusion protein to viable or -/- apoptotic Jurkat T cells. Cells were incubated with SIRPα- binding of apoptotic CD47 thymocytes Fc, followed by a PE-conjugated anti-human Fc-specific to non-activated macrophages. secondary antibody, and analysis by flow cytometry.

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 , 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 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 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

33

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

34 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 .

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

35 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).

36

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.

37

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,

38

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.

39

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