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The phagocyte inhibitory receptor sirpα in the immune system
Álvarez Zárate, J.
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Download date:23 Sep 2021 THE PHAGOCYTE INHIBITORY RECEPTOR SIRP
THE PHAGOCYTE INHIBITORY RECEPTOR SIRPα IN THE IMMUNE SYSTEM α IN THE IMMUNE SYSTEM JULIÁN ÁLVAREZ ZÁRATE 2014 ÁLVAREZ ZÁRATE JULIÁN
JULIÁN ÁLVAREZ ZÁRATE 2014 The phagocyte inhibitory receptor SIRPα in the immune system
Julián Álvarez Zárate
2014 © Julián Álvarez Zárate, Amsterdam, The Netherlands. Feel free to reproduce or photocopy any part of this thesis, but remember to acknowledge the author. Cover illustration done by Israel Páez. Printed by Off Page, Amsterdam. Printing of this thesis was financially supported by Sanquin research, Academic Medical Center (AMC) and the University of Amsterdam. ISBN: 978-94-6182-495-0 The phagocyte inhibitory receptor SIRPα in the immune system
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op donderdag 6 november 2014, te 14:00 uur
door
Julián Álvarez Zárate
geboren te Buenos Aires, Argentinië Promotiecommissie
Promotor: Prof. Dr. T.W. Kuijpers
Co-Promotor: Dr. T.K. van den Berg
Overige leden: Prof. dr. P.L. Hordijk Prof. dr. L. Meyaard Prof. dr. A.J. Verhoeven Dr. I. Maridonneau-Parini Dr. M.A. Nolte
Faculteit der Geneeskunde CONTENTS
Chapter 1 Introduction 7
Chapter 2 SIRPα controls the activity of the phagocyte NADPH oxidase 27 by restricting the expression of gp91phox
Chapter 3 Engagement of SIRPα inhibits growth and induces programmed 51 cell death in acute myeloid leukemia cells
Chapter 4 The Variola virus CD47 homologue A41L (vCD47) mediates 79 interactions with human SIRP family members on myeloid cells
Chapter 5 Regulation of phagocyte migration by signal regulatory 97 protein-alpha signaling
Chapter 6 Disorganized podosomes and impaired adhesion and 113 chemotaxis in macrophages from LADIII patients
Chapter 7 Summary and general discussion 133
Chapter 8 Acknowledgements 145
Introduction 1
The immune system 1 Infectious and often pathogenic organisms including viruses, bacteria, fungi and parasites are present in our environment in great amount and diversity. Like most if not all multicellular organisms humans are equipped with an immune system, which has the aim to recognize and eliminate these pathogens, while causing minimal damage to the host. The first primitive immune systems arose hundreds of millions of years ago, early in the evolution of multicellular organisms (1). The most ancient form of immunity is called innate immunity. The term innate refers to the fact that the relevant proteins involved in recognition and execution of the immune response are completely encoded within the genome. In mammals the innate immune system comprises an elaborate system of mucosal barriers, soluble mediators, and specialized cells, including phagocytes and NK cells, which collectively provide a first line of defense against pathogens (2). In addition to the innate immune system, adaptive immunity appeared later in evolution in early vertebrates and added a whole new level of complexity. This type of immunity is acquired during the lifetime of the organism and is shaped by its encounters with pathogens. Adaptive immunity is characterized by the presence of specialized antigen receptors, like the T and B cell antigen receptors (3). These antigen receptors, which are present in all jawed vertebrates, are created by a unique process of V(D)J-gene rearrangement, in which the antigen recognition domains of these receptors are assembled from a diverse number of gene segments partly encoded in the genome, which is further diversified by additional processes (4). This creates a virtually unlimited repertoire of antigen receptors, allowing the host to recognize and distinguish an impressive variety of pathogens. The T and B lymphocyte antigen receptors are expressed in a clonal fashion and when a particular lymphocyte recognizes an antigen that particular clonal population will become activated, proliferate and differentiate to give rise to progeny with the same specificity that is highly equipped and specialized for fighting that particular infection (5). Importantly, it will create long-term memory lymphocytes that can fight possible subsequent infections more efficiently, and thereby adjust the immune system of the host to its pathogenic environment.
Phagocytes Macrophages and granulocytes, collectively termed phagocytes, provide a first line of defense against invading pathogens. Phagocytes have the capacity to directly trace, internalize and kill pathogens as well as to contribute to the generation and effectuation of the adaptive immune response as well (6). Particularly among macrophages there is a great deal of heterogeneity in terms of morphology, phenotype and function. Apart from roles in host defense many of the resident tissue macrophages fulfill specialized homeostatic functions, which are as yet only partly understood (7). One of the most common homeostatic functions of macrophages involves the clearance of dead cells and cell remnants, which forms a continuous aspect of tissue renewal and repair in the body, but there are also highly specialized roles dedicated to particular tissue function (8). Whereas essentially all phagocytes are hematopoietic cells that derive from the common myeloid progenitor in the bone marrow or, depending on
9 developmental stage of the organism, also other blood forming tissues, such as the fetal liver or yolk sac, some of the resident tissue macrophages appear to form local pools of self- replenishing cells (8). However, most of the circulating phagocytes, including monocytes and granulocytes, directly emerge from the bone marrow (9). Both of these cell populations play particularly important roles during infection and other inflammatory diseases, when they can be recruited to the site of inflammation. Granulocytes, also known as polymorphonuclear granulocytes, include eosinophils, basophils, and neutrophils. Granulocytes are relatively short lived cells. Neutrophils for instance, which constitute the major subset of granulocytes making up 70% of all leukocytes in humans, survive for 5-6 days (10) leading to a turnover of ~109 cells/ kg per day, which may therefore amount to an impressive number of ~1011 cells that have to be generated by the bone marrow of an human individual every day (11). Upon inflammation, neutrophils can infiltrate tissues within hours and they are highly effective in the recognition, phagocytosis and intracellular killing of pathogens, in particular bacteria and fungi. The later is best illustrated by patients with (rare) genetic defects in neutrophil formation or function, which display and enhanced susceptibility to bacterial and fungal infections. Some of the major properties and effector functions of phagocytes in host defense are outlined in Figure 1 and these will be further outlined in the sections below. Closely related to phagocytes in terms of origin are the myeloid-type of dendritic cells (as opposed to dendritic cells that are from lymphoid origin). Dendritic cells (DC) are specialized in sensing and sampling antigens, derived amongst others from pathogenic microorganisms,
Figure 1. Properties and effector functions of phagocytes in homeostasis and host defense. Extravasation, migration and pathogen recognition are basic properties of phagocytes which help these fight pathogens by means of phagocytosis and killing through different mechanisms.
10 that they transport to the lymphoid tissues, such as the spleen or lymph nodes in order to activate T lymphocytes. Upon activation T cells can either differentiate into T helper cells that 1 provide help to B lymphocytes, which in turn will produce antibodies directed to the microbe in question, or they develop into cytotoxic T cells that mediate the elimination of virus-infected cells, or they become regulatory T cells that control the responses of the other T cells. These adaptive immune responses are associated with the formation of long-lived memory cells can come into action quickly and can effectively protect against a second infection, a principle that is used in vaccination. After the adaptive responses are established, which can take a week or more, phagocytes, including macrophages or granulocytes, again play a critical role in, for instance, the antibody-dependent elimination of extracellular microbes or infected cells (5). Thus, phagocytes act at essentially all stages of the immune response.
2.1 Migration Migration of phagocytes is of critical importance during the process of inflammation because it allows the cells to get to the site of infection and/or tissue repair. In order for granulocytes and monocytes to enter the inflammatory lesion they must generally first exit the blood circulation, a process known as extravasation. To do so phagocytes use a series of molecules including selectins and integrins to interact with endothelial cells that cover the luminal side of the vasculature. Selectins and integrins act, respectively, to mediate rolling and subsequent firm adhesion to the endothelial cells. Once firmly adhered they spread over the endothelial cell and from a pseudopodia that help the leukocyte to “squeeze” itself through the endothelial layer, a process known as diapedesis (12). Next, the basement membrane composed of dense extracellular matrix material is passed, in a process that might involve proteolytic degradation as well (13). After extravasation phagocytes employ gradients of chemokines or other chemotactic substances to guide them to the site of infection or injury (14). Within the interstitial tissue different types of interstitial migration can occur depending on the architecture of the matrix that the phagocyte encounters and the nature of the phagocyte itself. Whereas all phagocytes can migrate very fast and efficiently using amoeboid migration in porous matrices, some phagocytes, like macrophages or dendritic cells, can also migrate in very dense extracellular matrixes using the mesenchymal mode of migration (15). The latter require specialized adhesion structures know as podosomes that mediate the extracellular delivery of proteases that can degrade the matrix to create a passage for the phagocyte (16). Coordinated remodeling of the cellular actin cytoskeleton generates forces for contraction and relaxation, which are obviously critical for all stages and types of migration (17). Although this is known to involve a great number of cellular proteins, the exact way in which leukocyte motility is coordinated and regulated is only partly understood.
2.2 Pathogen recognition and inflammatory mediator production Phagocytes recognize pathogens by virtue of so called pattern recognition receptors (PRRs), which may be expressed on the cell surface or in the cytosol for detection of extracellular and intracellular pathogens respectively (18). Several of these PPRs recognize pathogen associated
11 molecular patterns (PAMPs), which upon ligation are able to activate the phagocyte and initiate a signaling cascade that leads to the production of inflammatory mediators or activation of other effector functions on the phagocyte (19). Among the best characterized PRRs are the Toll-like receptors (TLRs) (20), which are able to identify a variety of microbial ligands (e.g. LPS, flagellin, zymosan), the NOD-like receptors (NLR), that recognize amongst other things peptidoglycans from bacteria (21) and the lectins, which recognize carbohydrate moieties exposed on the surface of pathogens. PRRs can also be receptors for self ligands (e.g. mitochondria, heat shock proteins, heme) (22) which are collectively known as danger-associated molecular patterns (DAMPs). These DAMP receptors are available to the phagocyte when there is an injury or an insult associated with necrosis or other tissue damage that needs host defense and repair. One of the major responses that occur upon triggering of PRRs on phagocytes is the production of cytokines, chemokines, and other (pro) inflammatory mediators. These play a central role in recruiting and activating other immune cells and thus act as an important amplifiers and regulators of inflammatory responses (14). In addition, there are also anti-inflammatory mediators that are important for controlling the inflammatory response and to prevent excessive responses that can be harmful to the body and cause autoimmunity. Apart from these types of PRRs that mediate and coordinate inflammatory responses phagocytes can recognize microbes via opsonins, which are soluble molecules generated by the host that bind pathogens. By binding to opsonin receptors on the phagocyte these molecules promote recognition and phagocytosis, and trigger other effector functions (23;24). Examples of opsonins include, complement fragments, which bind to phagocyte complement receptors such as the CD11b/CD18 integrin (also know as complement receptor 3, CR3) and the antibodies of the adaptive immune system that bind to a variety of Fc receptors, of which many are also abundantly expressed by phagocytes.
2.3. Phagocytosis Phagocytosis literally means “the process of engulfing by a cell”. Is a specific type of endocytosis that involves vesicular internalization of solid particles, generally defined as having a size of ~1 µm or more, and constitutes a pivotal function within the immune system in which phagocytes ingest pathogens and death cells, such as apoptotic cells, or other host-derived waste material, for intracellular killing and/or lysosomal degradation. The process is initiated by recognition events mediated by specific receptors on the phagocyte plasma membrane that interact with ligands on the surface of the target particle. In the case of phagocytosis of pathogens there are a variety of receptors that can trigger phagocytosis. This includes opsonic Fc-receptors, which recognize antibody bound particles, and complement receptors, which bind to complement proteins (e.g. CD11b/CD18) or other non-opsonic receptors like scavenger receptors and lectins, which interact directly with target structures (24). In the context of phagocytosis of apoptotic cells other less well characterized receptors bind to ‘eat-me’ signals exposed on aged or dying cells (25). Phagocytosis requires remodeling of the phagocyte actin cytoskeleton enabling internalization of the particle into an intracellular compartment termed the phagosome. Phagosome formation involves the fusion with lysosomal vesicles that deliver a variety of hydrolytic enzymes into the phagosome and are responsible for the
12 compartmentalized degradation of the target (26). This ensures efficient killing of microbes and recycling of biological material with minimal damage to the surrounding tissue. 1
2.4. Killing The intracellular, and perhaps under certain conditions also extracellular killing by phagocytes, and in particular that by neutrophilic granulocytes, which are generally most important and also best characterized in this respect, depends on at least two important effector mechanisms. The first is the formation of reactive oxygen species (ROS) derived from superoxide, which is generated by electron transfer from cytoplasmic NADPH to intraphagosomal or extracellular oxygen by a protein complex termed the phagocyte NADPH oxidase. This complex is formed by gp91phox and p22phox, which are anchored to the membrane, and by p47phox, p67phox, p40phox and Rac1 or Rac2 which are cytoplasmic proteins that upon activation of the phagocyte translocate to the membrane to form a fully functional NADPH oxidase complex (27). Defects in gp91phox, p22phox, p47phox p67phox or p40phox result in chronic granulomatousis disease (CGD) (28), which causes a higher susceptibility to bacterial and fungal infections, demonstrating the importance of ROS generation on the host defense against pathogens. The other main antimicrobial function that neutrophils use is the release of hydrolases (particularly proteases), from preformed and stored granules, into the phagosomal or the extracellular compartment (29). The neutrophil azurophilic granules in particular contain serine proteases like elastase and others that are instrumental in pathogen killing (30). The activation of phagocytes i.e. the cellular pathways that lead to the activation of the various cellular effector functions, have been intensively studied . This has lead to a great deal of understanding and identification of molecules and signaling pathways involved on neutrophil and macrophage activation. These include e.g. the Toll-like receptors (TLR) that modulate e.g. cytokine production via the NFκB and other pathways, Fc receptors and other receptors with immune receptor tyrosine-based activating motifs (ITAMs) that signal amongst other things via the pivotal tyrosine kinase Syk, which in turn is responsible for activating a variety of pathways that induce various effector functions, such as phagocytosis and killing. However, inhibition and control over these processes is of primordial importance to prevent excessive activation that would cause damage to the host and perhaps even leading to autoimmune or autoinflammatory disease. Although clearly less well characterized than the activating pathways, inhibitory receptors often equipped with cytoplasmic immune receptor tyrosine-based inhibitory motifs (ITIMs) that provide them with inhibitory capacity have been shown to be instrumental in limiting excessive phagocyte and other immune cell type activation (31). The cytoplasmic ITIMs of these inhibitory receptors recruit and activate tyrosine phosphatases like SHP-1 and SHP-2 that are able to dephosphorylate various signaling proteins thereby homeostatically controlling or balancing phagocyte activation. Although a number of such inhibitory receptors, which bind to a range of (extra)cellular ligands have been described on phagocytes, including SIRPα, CD200R, CD300, LAIR, several siglec and ILT family members (32), the individual contributions of these inhibitory receptor systems in relation to the specialized functions of phagocytes are not well understood at all currently. In the next section I will discuss the role of SIRPα in particular, which is the inhibitory of primary interest in the studies described in this thesis.
13 Signal regulatory protein alpha The SIRPα glycoprotein receptor was identified independently by a number of laboratories (33-36). While its was first characterized to control receptor and non-receptor tyrosine kinase signaling in non-hematopoietic tumor cell lines such as fibroblasts and epithelial cells, were it is apparently expressed as a consequence of transformation and/or in vitro culture, it was later realized to be selectively expressed in myeloid cells and neurons in vivo (35).
3.1. Genetics, evolution, family relationships and ligand specificity SIRPα is actually the prototypic member of a larger multigene family of Signal Regulatory Proteins (SIRPs). SIRPs form a family of immunoreceptor proteins encoded by a cluster of genes on chromosome 20p13 in humans (37). SIRP family members have been identified in mammals, birds and reptiles and all of the genes and pseudogenes within these clusters have been generated by duplication events from SIRPα, which is the founding member of the family (see below). SIRPs form a family of so called ‘paired’ receptors, with members of the family sharing a high level of similarity in their extracellular domains (Figure 2), but having the capacity to generate either inhibitory or activating intracellular signals. Whereas SIRPα (also known as CD172a, SHPS-1, BIT, MyD-1, MFR), contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its intracellular domain, other SIRPs, such as the less well characterized SIRPβ1 (CD172b) or SIRPβ2, may transduce activating signals through the associated ITAM-containing adapter molecule DAP12 (38). SIRPγ (CD172g) is a member without any known signaling motifs (39). Due
Figure 2. SIRP family. Schematic representation of the SIRP protein family members. Blue and red ovals represent V- and C1-set Ig domains respectively. The Y letters represent ITIM tyrosine residues. The “+” represent positive charged lysine residues in the transmembrane regions of SIRPβ1 and SIRPβ2, which are essential for association with the ITAM-containing and generally activating adaptor molecule DAP12.
14 to critical roles in immunity and infection, which provide a strong driving force for selection and therefore evolutionary diversification, many paired receptor families display a high level of gene 1 diversification, including variable gene numbers among species and considerable sequence variation of a certain gene with a given species (i.e. polymorphic variation (37;40;41) (Zarate JA et al, unpublished). Whereas SIRPα, SIRPβ1 and SIRPγ show a similar overall organization of the extracellular domain and a high level of homology, the SIRPβ2 and the putatively soluble family member SIRPδ are more distantly related. The SIRP family exhibits a considerable variability across species but in all mammalian species characterized until now a SIRPα ortholog with a preserved structure of 3 extracellular domains and a long cytoplasmic tail containing ITIMs is present. The other members of the family tend to differ in number and structure clearly identifying SIRPα as the prototypic and primordial member of the family. Why is SIRPα so conserved among species whereas the other members are not? This might have to do with differences in function: SIRPα is an inhibitory protein involved in homeostatic regulation of cell function through interaction with an endogenous ligand, i.e. CD47, whereas the other SIRPs have arisen by gene duplication, mutation and gene loss (pseudogenes) from the primordial SIRPα. Both SIRPα and, with ~10-fold lower affinity, SIRPγ bind the broadly expressed CD47 surface molecule(42)-(39;43;44) originally identified as integrin-associated protein, but the potentially activating members SIRPβ1 and SIRPβ2 (Zarate JA and Van den Berg, unpublished) (45) do not. Interestingly, there appear to be a considerable number of polymorphic variants of SIRPα in different ethnic populations, including Africans and Asians, with as many as 10 different SIRPα variants identified in 39 individuals investigated (40). Although this is quite an impressive frequency this is not completely unprecedented, since many inhibitory immunoreceptors that bind highly polymorphic MHC class I antigens like e.g. KIRs and Ly-49 also show a high degree of polymorphic variation. Nevertheless, the difference is that the ligand for SIRPα, CD47, is in contrary to the highly polymorphic MHC molecules a very well conserved protein. Moreover, the major polymorphic variants in the Caucasian population (Zhao et al, submitted), SIRPα1 and SIRPαBIT, which form the two extremes within the spectrum of differences among the SIRPα polymorphisms, show identical affinities for CD47 (45) and similar levels of regulation of phagocytic function (Zhao et al., submitted). This is consistent with the observation that the polymorphic amino acid residues occur primarily in regions of the N-terminal ligand binding Ig-like domain of SIRPα that are just flanking the CD47 binding site. It is also in agreement that CD47-SIRPα interactions serve important homeostatic functions in the immune and central nervous systems. It has been suggested that the evolutionary pressure to drive such level of polymorphisms might be caused by pathogens targeting SIRPα (further discussed in chapter 4), which they might do so for the purpose of inhibiting SIRPα-dependent phagocyte effector functions, thereby improving their chances of survival in the host. It seems reasonable to assume that such mechanisms, which are based on host-pathogen interactions and competition generate a strong driving force to promote diversity in inhibitory receptors and probably also the emergence of activating members, which would function in an inverse fashion, namely to act as receptors for such pathogens (45;46). Of interest in this context, poxviruses express a homologue of CD47, termed vCD47, which might perhaps constitute a ligand expressed on
15 poxvirus-infected cells for SIRPα, and/or any of the other SIRP family members. Interestingly, in vivo studies in rabbits using a Myxoma virus deficient for this vCD47 have shown that it acts as a virulence factor and also to suppress macrophage activation (47). However, no evidence for interactions of vCD47 with SIRP family members have so far been reported. A number of structural aspects of the SIRP family members, including the presence of C1 domains, the presence of a J-like sequence in the ligand binding V-set Ig-like domain, its mode of interaction with CD47 through 3 loops of this V-domain, and its signaling through ITAM-containing adapter proteins are all reminiscent of T cell receptors (TcR). T and B cell receptors are, however, unique in their capacity to be subject to V(D)J-rearrangement which, creates the diversity required to recognize the many different antigens in our environment. Antigen receptor rearrangement occurs in all jawed vertebrates and it is believed that during evolution this capacity for rearrangement emerged by the insertion of a transposon into the V-domain of a non-rearranging primordial. The structural similarities between SIRP and TcR indicates that a SIRP-like gene in such an early vertebrate species may well have been the target for such a transposition event, which puts the SIRP family in a key position during the evolution of the adaptive immune system.
3.2 SIRPα signaling SIRPα is highly expressed in myeloid cells, including granulocytes, macrophages and myeloid DC, as well as neurons whereas its ligand CD47 is found virtually on all cells in the body. The CD47-
SIRPα interaction, which involves the NH2 terminal V-set Ig-like domain of SIRPα and the single Ig-like domain of CD47 (45) leads to bidirectional signaling events downstream of each of the two receptors (48). SIRPα signaling is executed through its intracellular domain. This contains 4 tyrosines that form 2 typical ITIM motifs, which upon phosphorylation of the tyrosines recruit the cytosolic tyrosine phosphatases SHP-1 and SHP-2 (49). Binding of the SH2 domains of these phosphatases unmasks their activity, which is otherwise auto-inhibited, and allows them to dephosphorylate proximal substrates, and thereby to affect downstream signaling pathways, generally in a negative fashion (Figure 3). Although presumably very important the signaling through SHP-1 and SHP-2 may not be the only way SIRPα can transduce signals. Biochemical experiments have actually suggested SIRPα to function as a scaffold protein for several other signaling proteins (36), and although for most of these proteins there is no evidence for a functional role in SIRPα signaling, the Src Kinase-associated phosphoprotein 2 (SKAP2) forms an exception, because it has recently been shown to be relevant in the context of macrophage integrin signaling and cytoskeletal remodeling signaling downstream of SIRPα (50). Furthermore, the two proline-rich regions found between the tyrosines in the ITIMs could potentially bind to SH3 domain-containing proteins that may also be instrumental in SIRPα signal transduction. The resulting intracellular signaling events have been reported to regulate a number of phagocyte functions and these will be outlined in the following sections. However, as indicated above ligation of CD47 by SIRPα can lead to signaling as well, making this a bidirectional signaling system (51). CD47 has a tetraspanning transmembrane region that can associate with cytoplasmic heterotrimeric Gi proteins, and in cis, i.e. in the horizontal plane of the membrane, with certain integrins, such as β3 integrins, and can regulate diverse downstream functions as well.
16 1
Figure 3. SIRPα signal transduction upon CD47 binding. SIRPα–CD47 interactions trigger the recruitment of phosphatases like SHP1 and SHP2 to the ITIMs of SIRPα resulting in their activation and the dephosphorylation of different target substrates which in turn regulate cell functions. The oval with the question mark represents other possible adaptor molecules that could interact with the cytoplasmic tail of SIRPα and contribute to signal transduction (50)
3.3. Regulation of phagocyte function by SIRPα 3.3.1. Adhesion and migration Adhesion to cells and extracellular matrices and migration are essential for phagocytes to extravasate and, subsequently, to enter and patrol tissues and inflammatory sites, and to interact with host cells and pathogens. Integrins are of pivotal importance in all of these processes because they are the adhesion molecules that provide the anchoring points at the cell surface of phagocytes and other immune cells or the extracellular matrix needed to translate the tracking forces generated by the cytoskeleton into cellular motility or spreading (12). In fibroblasts SHP2 has been shown to translocate to adhesion structures upon integrin engagement and to bind to SIRPα (33). Upon integrin engagement Src PTKs phosphorylate the tyrosines in SIRPα cytoplasmic tail and this leads to recruitment and activation of SHP2 (52). Furthermore, a SIRPα mutant mouse lacking the cytoplasmic tail has been generated (53) and embryonic fibroblasts from these mutant mice were found to display enhanced actin stress fiber formation and focal adhesion structure formation. Moreover, the migration of these cells was impaired (53) possibly involving a role for RhoA acting downstream of SIRPα. Similar to integrins SIRPα ligation on myeloid cells by CD47 can promote phosphorylation of the SIRPα ITIMs (54), but integrin-mediated adhesion is essential for this (55). In the context of transendothelial migration several studies have shown a role in vitro for CD47-SIRPα interactions. Blocking antibodies against SIRPα or recombinant CD47 protein decreased the ability of human neutrophils to transmigrate in a transwell assay (56), and another study performed with rat monocytes showed similar results with monocytes transmigrating across a monolayer of brain endothelium (57). The latter study suggested that in particular the diapedesis step of the extravasation process involved CD47-SIRPα interactions and also CD47 signaling, perhaps to open the endothelial junctions, but it is not known whether also signaling through SIRPα the phagocyte
17 was required. Furthermore, it is not known whether CD47-SIRPα interactions play a role during the in vivo migration of phagocytes. Studies to address these issues are described in chapter 5. While there are many questions with respect to the migration of granulocytes, monocytes and macrophages, more knowledge is available about the role of CD47 and SIRPα in the migration of dendritic cells. Treatment with blocking anti-SIRPα Abs or genetic ablation has been shown to result in reduced migration of Langerhans cells to the draining lymph nodes in mice (58). This is interesting in the context of infection, were deficient DC functions, like antigen presentation and induction of T cells responses, have been found to be deficient on the SIRPα mutant mouse (59). However, whether the defect in DC migration in SIRPα-mutant mice completely accounts for the impaired T cells responses is not clear, because other relevant aspects such as e.g. formation of the immunological synapse may also be dependent on CD47-SIRPα interactions. Related to this last possibility we have recently observed that synapse killer formation between neutrophils and antibody-opsonized target cancer cells during antibody-dependent cytotoxicity (ADCC), which is critically dependent on so called inside-out activation of the CD11b/CD18 integrin by the integrin-binding cytoplasmic protein kindlin3, is limited by CD47-SIRPα interactions (Matlung M., Zhao X. et al., unpublished). Interestingly, experiments with neutrophils from patients with Leukocyte Adhesion Deficiency type III syndrome, which have mutations in the FERMT3 gene and consequently lack the kindlin3 protein, indicated that SIRPα signaling controls this kindlin3- dependent killer synapse formation. The latter suggested a potential role for SIRPα in neutrophil integrin regulation and also that kindlin3 might actually function as a target downstream of SIRPα for regulations of phagocyte function. Kindlin3 deficiency in patients leads to a typical leukocyte and platelet integrin adhesion deficiency, because it precludes the β2- and β3- integrins, of respectively leukocytes and platelets, to obtain a high affinity ligand binding conformation. As a result patients have an enhanced numbers of neutrophils and other leukocytes in the circulation, as well as a bleeding tendency as a result of a defect in platelet aggregation. The defect in neutrophil extravasation can be explained by a lack of integrin-mediated firm adhesion to endothelial cells (60), but integrins and kindlin3 could potentially also regulate phagocyte migration at other levels. There is evidence, for instance, that integrins control the mesenchymal interstitial migration of fibroblasts, myoblasts, single endothelial cells or sarcoma cells among others (61). Since macrophages, but not neutrophils, are also able to perform mesenchymal migration it was of interest to study the function of kindlin3 in macrophages. Studies to describe the role of kindlin3 in macrophage migration are described in chapter 6.
3.3.2. Host Cell Phagocytosis One of the best studied physiological functions regulated by the SIRPα-CD47 interaction is the homeostasis of red blood cells (RBC) and platelets. Evidence for this was originally obtained by transfusing CD47-deficient RBC to wild-type mice and monitoring their clearance (62). Whereas a RBC of a wild-type mouse have a normal survival of around 60-80 days, the CD47- deficient cells were cleared from circulation within hours, suggesting that CD47 functions as a potent ‘don’t-eat-me’ signal. Further experiments (62) indicated that this was because of a lack of SIRPα signaling in spleen macrophages. CD47 knock-out (KO) and SIRPα-mutant mice,
18 which lack the cytoplasmic tail of the receptor and therefore its signaling capacity, show only a relatively mild anemia (63;64)(Zarate JA et al. unpublished). Of interest, subsequent studies 1 showed that this apparent discrepancy can be explained by the fact that macrophages are one or another way instructed by CD47 expressed on non-hematopoietic cells with respect to their capacity to respond to SIRPα triggering (65). Such a mechanism is reminiscent of the ‘licensing’ of natural killer cells for the killing of MHC class I-deficient targets (66). Recently it has been shown that CD47-SIRPα interactions might regulate RBC clearance in a more complicated fashion than what was originally established. Burger et. all found that on human, RBC ageing is related to a conformational change on CD47 that transforms the molecule from a “don’t-eat-me” into an “eat-me” configuration and that this conformational change occurs during RBC aging (67). Apart from this well characterized anemia these mice have also been shown to be mildly thrombocytopenic (Zarate JA et al. unpublished) (64). Since there appear not to be intrinsic defects in megakaryopoiesis in these mice, these findings are consistent with an increased level of platelet clearance too, as has been reported now for many other hematopoietic cells, including healthy and leukemic hematopoietic stem cells and memory T lymphocytes now as well (68-70).
3.3.3. Transplantation and Xenotransplantation Organ transplantation is the only solution for survival for many patients suffering from irreversible organ damage. Unfortunately, matched donors are not always readily available and many patients die while on the transplantation waiting list. One possible alternative is xenotransplantation with e.g. pig organs. Clearly, graft-versus-host reactions are a big concern and constitute the main difficulty to bypass in xenotransplantation. In this context Takenaka et al. discovered that SIRPα was responsible for the superior ability of the NOD/SCID mouse strain to accept human xenotransplants (40). It is known that CD47-SIRPα-CD47 interactions are not well conserved across species and this incompatibility of the CD47-SIRPα interaction apparently hampers xenotransplantation (71;72). However, the NOD mouse SIRPα allele was found to bind to human CD47 with higher affinity than alleles expressed by other commonly used mouse strains, which have only very low affinity for human CD47. This promoted acceptance of the human graft by inhibiting phagocytosis by macrophages. This showed that CD47-SIRPα interactions regulate not only phagocytosis of host cells but also form an important barrier for xenotransplantation. Subsequent studies using mice that express a human SIRPα transgene and human hematopoietic cells expressing a mouse CD47 molecule confirmed this concept. Finally, formal proof that the NOD SIRPα is both essential as well as sufficient for human graft acceptance came from knock in studies with the NOD SIRPα allele into other non-permissive mouse strains (68). Clearly, this discovery has also improved humanized mouse models to study disease.
3.3.4. Cancer As described above CD47 act as a “don’t eat me” signal to prevent phagocytosis of host cells by macrophages. This makes the CD47-SIRPα interactions interesting to in the context of cancer cell clearance. It has been shown that many cancer cells of different lineages express high levels
19 of CD47 and that cancer stem cells may express particularly high levels (70). Consistent with this, CD47 can actually serve as a prognostics factor, with cancers expressing high levels of the molecule having worse prognosis (73). Indeed, it seems that CD47 on cancer protects them from clearance by macrophages and thereby contribute to their survival in vivo (70). Several studies have exploited this concept and interfered with the CD47-SIRPα interaction having successfully reduced engraftment of cancer cells or in some cases even eliminated pre-established leukemia in mouse xenograft tumor models (70;73;74) (75). However, these studies used intact anti-CD47 monoclonal antibodies and there is controversy on whether disturbance of the CD47-SIRPα interaction or opsonization of the cancer cells by antibodies is responsible for tumor cell clearance (76). However, it is clear from experiments using SIRPα mutant mice and from recent studies using a recombinant affinity-enhanced SIRPα protein in mouse models that CD47-SIRPα interactions or SIRPα signaling can potentiate the effects of all the major cancer therapeutic antibodies used in human patients (41;77). In addition, there is evidence that CD47 can directly convey pro-apoptotic signals into certain cancer cells (78). In chapter 3 studies are described to show that the latter might also be the case also for SIRPα in acute myeloid leukemic cells.
3.3.5. Autoimmunity SIRPα has essentially been characterized as an inhibitory receptor that homeostatically counterbalances cellular immune effector functions. From this perspective it is interesting to study its role on autoimmunity. Spontaneously, both SIRPα mutant and CD47 KO mice develop mild anemia and thrombocytopenia as a result of the enhanced clearance of these hematopoietic cells. Therefore, it is not surprising that CD47 KO mice show lethal autoimmune hemolytic anemia (AIHA) (63) or idiopathic trombocytopenic purpura (ITP) upon injection with antibodies to respectively red cells or platelets. However, these are passive models in which only the effects of autoantibodies are studied, but it is not known whether in AIHA and ITP also the formation of autoantibodies is promoted by interference with CD47-SIRPα interactions. In fact, it appears that in many T cell-mediated autoimmune models, such as encephalomyelitis (79) or collagen induced arthritis (80), SIRPα-mutant mice are resistant to the establishment of disease. The reason for this perhaps unexpected resistance may be that CD47-SIRPα interactions are necessary for optimal DC migration and antigen presentation by these cells to T cells (79).
3.3.6. Infection Phagocytes are essential in the host defense against pathogens. The role of SIRPα regulating killing of pathogens by phagocytes as a first line of defense has not been extensively studied. Some in vitro studies have suggested that SIRPα on macrophages can negatively regulate TLR3 and TLR4 signaling (81;82) and suppress intracellular killing of bacteria (83). However, it is important to realize that all of these studies have been done with either shRNA or siRNA targeting with virus and this may affect macrophage activation in a non-specific fashion through the triggering of ‘danger’ pathways. Therefore, additional evidence is certainly required to support or contradict these claims. A recent study has shown that SIRPα is essential for controlling Salmonella infection in vivo (59). The results of the latter study support a concept
20 in which the lack of SIRPα signaling in dendritic cells results in less efficient antigen processing and presentation, which in vivo leads to poor Salmonella-specific CD4 T cell and antibody 1 responses. Whether the regulation of granulocyte and macrophage anti-microbial activities are somehow impaired and may also contribute to the inability to clear bacterial or other types of infection is not known. Studies to further address these issues are described in chapter 2.
Scope of the thesis Phagocytes play a central role in the host defense against pathogens, by virtue of amongst other things their ability to recognize and destroy them. These processes have to be tightly controlled to prevent unwanted inflammation that could harm the host. From previous studies it has become clear the interactions between SIRPα expressed on phagocytes and CD47 on other cells is involved in the homeostatic control of immune effector functions, such as e.g. phagocytosis, against host cells (Figure 3). The research presented in this thesis aimed to explore whether SIRPα was also involved in the regulation of other previously unidentified aspects of phagocyte immunity. In chapter 2 we demonstrate that SIRPα specifically regulates the phagocyte NADPH oxidase activity, an essential phagocyte effector function in the killing of bacteria and fungi. This involves both interactions between CD47 and SIRPα, as well as SIRPα signaling through the ITIM motifs and downstream signaling to control expression levels of the gp91phox protein, which comprises the catalytically active component of the NADPH oxidase complex. As a result phagocytes from SIRPα-mutant mice have a higher NADPH oxidase capacity and this could potentially confer enhanced protection against microbial infection. In chapter 3 we have studied the expression of SIRPα in various subtypes of acute myeloid leukemia (AML), a malignancy of hematopoietic progenitor cells with variable levels of myeloid differentiation. Our results indicate that SIRPα expression is associated with AML subtype and the degree of myeloid differentiation of the AML. Furthermore, SIRPα expression is controlled by epigenetic mechanisms, and engagement of SIRPα can actually promote programmed cell death, identifying the receptor as a potential therapeutic target in AML. In chapter 4 we provide the first preliminary evidence that a CD47 homologue from the human poxvirus Variola, termed vCD47, interacts with SIRP family members. Although we have not been able to establish which family members are specifically involved, this provides the basis for understanding the reported immunoregulatory role for vCD47 during poxviral infection. Chapters 5 and 6 are dedicated to phagocyte migration. Here we describe experiments to evaluate the function of SIRPα signaling in phagocyte migration. Our findings are consistent with a rather subtle role of SIRPα signaling in the recruitment of granulocytes and monocytes to inflammatory sites and a role in amoeboid interstitial migration of macrophages (chapter 5). We also provide evidence that cytoplasmic protein kindlin-3, which is defective in patients Leukocyte Adhesion Deficiency (LAD)-III and critical for integrin affinity regulation plays a role in the formation of macrophage adhesion structures and migration. In the general discussion (chapter 7) we summarize and discuss these novel findings with respect to the role of SIRPα in phagocytes, and put them into broader context.
21 Reference List 1. Travis,J. (2009) Origins. On the origin of the 16. Schachtner,H., Calaminus,S.D., Thomas,S.G. and immune system. Science, 324, 580-582. Machesky,L.M. (2013) Podosomes in adhesion, 2. Muller,U., Vogel,P., Alber,G. and Schaub,G.A. migration, mechanosensing and matrix remodeling. (2008) The innate immune system of mammals Cytoskeleton (Hoboken. ). and insects. Contrib. Microbiol., 15, 21-44. 17. Parsons,J.T., Horwitz,A.R. and Schwartz,M.A. (2010) 3. Vivier,E. and Malissen,B. (2005) Innate and Cell adhesion: integrating cytoskeletal dynamics and adaptive immunity: specificities and signaling cellular tension. Nat. Rev. Mol. Cell Biol., 11, 633-643. hierarchies revisited. Nat. Immunol., 6, 17-21. 18. Takeuchi,O. and Akira,S. (2010) Pattern recognition 4. Grawunder,U., West,R.B. and Lieber,M.R. (1998) receptors and inflammation. Cell, 140, 805-820. Antigen receptor gene rearrangement. Curr. 19. Tang,D., Kang,R., Coyne,C.B., Zeh,H.J. and Lotze,M.T. Opin. Immunol., 10, 172-180. (2012) PAMPs and DAMPs: signal 0s that spur 5. Janeway CA Jr, Travers P,Walport M,et al (2001) autophagy and immunity. Immunol. Rev., 249, 158-175. Immunobiology: The Immune System in Health 20. Lee,C.C., Avalos,A.M. and Ploegh,H.L. (2012) and Disease. Accessory molecules for Toll-like receptors and 6. Kolaczkowska,E. and Kubes,P. (2013) Neutrophil their function. Nat. Rev. Immunol., 12, 168-179. recruitment and function in health and 21. Franchi,L., Warner,N., Viani,K. and Nunez,G. inflammation. Nat. Rev. Immunol., 13, 159-175. (2009) Function of Nod-like receptors in microbial 7. Wynn,T.A., Chawla,A. and Pollard,J.W. recognition and host defense. Immunol. Rev., 227, (2013) Macrophage biology in development, 106-128. homeostasis and disease. Nature, 496, 445-455. 22. Yu,L., Wang,L. and Chen,S. (2010) Endogenous 8. Lech,M., Grobmayr,R., Weidenbusch,M. and toll-like receptor ligands and their biological Anders,H.J. (2012) Tissues use resident dendritic significance. J. Cell Mol. Med., 14, 2592-2603. cells and macrophages to maintain homeostasis 23. Ehlers,M.R. (2000) CR3: a general purpose and to regain homeostasis upon tissue injury: adhesion-recognition receptor essential for the immunoregulatory role of changing tissue innate immunity. Microbes. Infect., 2, 289-294. environments. Mediators. Inflamm., 2012, 951390. 24. Aderem,A. and Underhill,D.M. (1999) Mechanisms 9. VOLKMAN,A. and GOWANS,J.L. (1965) THE of phagocytosis in macrophages. Annu. Rev. ORIGIN OF MACROPHAGES FROM BONE Immunol., 17, 593-623. MARROW IN THE RAT. Br. J. Exp. Pathol., 46, 62-70. 25. Ravichandran,K.S. (2010) Find-me and eat-me 10. Pillay,J., den Braber,I. Vrisekoop,N., Kwast,L.M., signals in apoptotic cell clearance: progress and de Boer,R.J., Borghans,J.A., Tesselaar,K. and conundrums. J. Exp. Med., 207, 1807-1817. Koenderman,L. (2010) In vivo labeling with 2H2O 26. Kinchen,J.M. and Ravichandran,K.S. (2008) reveals a human neutrophil lifespan of 5.4 days. Phagosome maturation: going through the acid Blood, 116, 625-627. test. Nat. Rev. Mol. Cell Biol., 9, 781-795. 11. von Vietinghoff,S. and Ley,K. (2008) Homeostatic 27. Babior,B.M. (1999) NADPH oxidase: an update. regulation of blood neutrophil counts. J. Blood, 93, 1464-1476. Immunol., 181, 5183-5188. 28. Roos,D., Kuhns,D.B., Maddalena,A., Roesler,J., 12. Ley,K., Laudanna,C., Cybulsky,M.I. and Lopez,J.A., Ariga,T., Avcin,T., de Boer.M., Nourshargh,S. (2007) Getting to the site of Bustamante,J., Condino-Neto,A. et al. (2010) inflammation: the leukocyte adhesion cascade Hematologically important mutations: X-linked updated. Nat. Rev. Immunol., 7, 678-689. chronic granulomatous disease (third update). 13. Schoumacher,M., Louvard,D. and Vignjevic,D. (2011) Blood Cells Mol. Dis., 45, 246-265. Cytoskeleton networks in basement membrane 29. Borregaard,N. and Cowland,J.B. (1997) Granules transmigration. Eur. J. Cell Biol., 90, 93-99. of the human neutrophilic polymorphonuclear 14. Moser,B. and Willimann,K. (2004) Chemokines: leukocyte. Blood, 89, 3503-3521. role in inflammation and immune surveillance. 30. Segal,A.W. (2005) How neutrophils kill microbes. Ann. Rheum. Dis., 63 Suppl 2, ii84-ii89. Annu. Rev. Immunol., 23, 197-223. 15. Le,Cabec., Van,Goethem,E., Guiet,R. and 31. Blank,U., Launay,P., Benhamou,M. and Maridonneau-Parini,I. (2011) [Phagocyte migration: Monteiro,R.C. (2009) Inhibitory ITAMs as novel an overview]. Med. Sci. (Paris), 27, 1112-1119. regulators of immunity. Immunol. Rev., 232, 59-71.
22 32. Ravetch,J.V. and Lanier,L.L. (2000) Immune 43. Jiang,P., Lagenaur,C.F. and Narayanan,V. (1999) inhibitory receptors. Science, 290, 84-89. Integrin-associated protein is a ligand for the P84 1 33. Fujioka,Y., Matozaki,T., Noguchi,T., Iwamatsu,A., neural adhesion molecule. J. Biol. Chem., 274, 559- Yamao,T., Takahashi,N., Tsuda,M., Takada,T. 562. and Kasuga,M. (1996) A novel membrane 44. Vernon-Wilson,E.F., Kee,W.J., Willis,A.C., glycoprotein, SHPS-1, that binds the SH2-domain- Barclay,A.N., Simmons,D.L. and Brown,M.H. (2000) containing protein tyrosine phosphatase SHP-2 in CD47 is a ligand for rat macrophage membrane response to mitogens and cell adhesion. Mol. Cell signal regulatory protein SIRP (OX41) and human Biol., 16, 6887-6899. SIRPalpha 1. Eur. J. Immunol., 30, 2130-2137. 34. Kharitonenkov,A., Chen,Z., Sures,I., Wang,H., 45. Hatherley,D., Graham,S.C., Turner,J., Harlos,K., Schilling,J. and Ullrich,A. (1997) A family of Stuart,D.I. and Barclay,A.N. (2008) Paired proteins that inhibit signalling through tyrosine receptor specificity explained by structures of kinase receptors. Nature, 386, 181-186. signal regulatory proteins alone and complexed 35. Adams,S., van der Laan,L.J., Vernon-Wilson,E., with CD47. Mol. Cell, 31, 266-277. Renardel de,L.C., Dopp,E.A., Dijkstra,C.D., 46. Akkaya,M. and Barclay,A.N. (2012) How do Simmons,D.L. and van den Berg,T.K. (1998) Signal- pathogens drive the evolution of paired regulatory protein is selectively expressed by myeloid receptors? Eur. J. Immunol.. and neuronal cells. J. Immunol., 161, 1853-1859. 47. Cameron,C.M., Barrett,J.W., Mann,M., Lucas,A. 36. Timms,J.F., Carlberg,K., Gu,H., Chen,H., and McFadden,G. (2005) Myxoma virus M128L is Kamatkar,S., Nadler,M.J., Rohrschneider,L.R. expressed as a cell surface CD47-like virulence and Neel,B.G. (1998) Identification of major factor that contributes to the downregulation of binding proteins and substrates for the SH2- macrophage activation in vivo. Virology, 337, 55-67. containing protein tyrosine phosphatase SHP-1 in 48. Matozaki,T., Murata,Y., Okazawa,H. and macrophages. Mol. Cell Biol., 18, 3838-3850. Ohnishi,H. (2009) Functions and molecular 37. van Beek,E.M., Cochrane,F., Barclay,A.N. and van mechanisms of the CD47-SIRPalpha signalling den Berg,T.K. (2005) Signal regulatory proteins in pathway. Trends Cell Biol., 19, 72-80. the immune system. J. Immunol., 175, 7781-7787. 49. Takada,T., Matozaki,T., Takeda,H., Fukunaga,K., 38. Dietrich,J., Cella,M., Seiffert,M., Buhring,H.J. Noguchi,T., Fujioka,Y., Okazaki,I., Tsuda,M., and Colonna,M. (2000) Cutting edge: signal- Yamao,T., Ochi,F. et al. (1998) Roles of the complex regulatory protein beta 1 is a DAP12-associated formation of SHPS-1 with SHP-2 in insulin- activating receptor expressed in myeloid cells. J. stimulated mitogen-activated protein kinase Immunol., 164, 9-12. activation. J. Biol. Chem., 273, 9234-9242. 39. Brooke,G., Holbrook,J.D., Brown,M.H. and 50. Alenghat,F.J., Baca,Q.J., Rubin,N.T., Pao,L.I., Barclay,A.N. (2004) Human lymphocytes interact Matozaki,T., Lowell,C.A., Golan,D.E., Neel,B.G. directly with CD47 through a novel member of and Swanson,K.D. (2012) Macrophages require the signal regulatory protein (SIRP) family. J. Skap2 and Sirpalpha for integrin-stimulated Immunol., 173, 2562-2570. cytoskeletal rearrangement. J. Cell Sci.. 40. Takenaka,K., Prasolava,T.K., Wang,J.C., Mortin- 51. Brown,E.J. and Frazier,W.A. (2001) Integrin- Toth,S.M., Khalouei,S., Gan,O.I., Dick,J.E. and associated protein (CD47) and its ligands. Trends Danska,J.S. (2007) Polymorphism in Sirpa Cell Biol., 11, 130-135. modulates engraftment of human hematopoietic 52. Oh,E.S., Gu,H., Saxton,T.M., Timms,J.F., stem cells. Nat. Immunol., 8, 1313-1323. Hausdorff,S., Frevert,E.U., Kahn,B.B., Pawson,T., 41. Weiskopf,K., Ring,A.M., Ho,C.C., Volkmer,J.P., Neel,B.G. and Thomas,S.M. (1999) Regulation of Levin,A.M., Volkmer,A.K., Ozkan,E., Fernhoff,N.B., early events in integrin signaling by protein tyrosine van de Rijn,M., Weissman,I.L. et al. (2013) Engineered phosphatase SHP-2. Mol. Cell Biol., 19, 3205-3215. SIRPalpha variants as immunotherapeutic adjuvants 53. Inagaki,K., Yamao,T., Noguchi,T., Matozaki,T., to anticancer antibodies. Science, 341, 88-91. Fukunaga,K., Takada,T., Hosooka,T., Akira,S. and 42. Seiffert,M., Cant,C., Chen,Z., Rappold,I., Brugger,W., Kasuga,M. (2000) SHPS-1 regulates integrin- Kanz,L., Brown,E.J., Ullrich,A. and Buhring,H.J. mediated cytoskeletal reorganization and cell (1999) Human signal-regulatory protein is expressed motility. EMBO J., 19, 6721-6731. on normal, but not on subsets of leukemic myeloid 54. van Beek,E.M., Zarate,J.A., van Bruggen.R., cells and mediates cellular adhesion involving its Schornagel,K., Tool,A.T., Matozaki,T., Kraal,G., counterreceptor CD47. Blood, 94, 3633-3643. Roos,D. and van den Berg,T.K. (2012) SIRPalpha
23 controls the activity of the phagocyte NADPH 65. Wang,H., Madariaga,M.L., Wang,S., Van,R.N., oxidase by restricting the expression of Oldenborg,P.A. and Yang,Y.G. (2007) Lack of gp91(phox). Cell Rep., 2, 748-755. CD47 on nonhematopoietic cells induces split 55. Johansen,M.L. and Brown,E.J. (2007) Dual macrophage tolerance to CD47null cells. Proc. regulation of SIRPalpha phosphorylation by Natl. Acad. Sci. U. S. A, 104, 13744-13749. integrins and CD47. J. Biol. Chem., 282, 24219-24230. 66. Orr,M.T., Murphy,W.J. and Lanier,L.L. (2010) 56. Liu,Y., Buhring,H.J., Zen,K., Burst,S.L., Schnell,F.J., ‘Unlicensed’ natural killer cells dominate the Williams,I.R. and Parkos,C.A. (2002) Signal response to cytomegalovirus infection. Nat. regulatory protein (SIRPalpha), a cellular ligand Immunol., 11, 321-327. for CD47, regulates neutrophil transmigration. J. 67. Burger,P., Hilarius-Stokman,P., de Korte,D., van Biol. Chem., 277, 10028-10036. den Berg,T.K. and van Bruggen,R. (2012) CD47 57. de Vries,H.E., Hendriks,J.J., Honing,H., De functions as a molecular switch for erythrocyte Lavalette,C.R., van der Pol,S.M., Hooijberg,E., phagocytosis. Blood, 119, 5512-5521. Dijkstra,C.D. and van den Berg,T.K. (2002) Signal- 68. Kuriyama,T., Takenaka,K., Kohno,K., Yamauchi,T., regulatory protein alpha-CD47 interactions are Daitoku,S., Yoshimoto,G., Kikushige,Y., Kishimoto,J., required for the transmigration of monocytes Abe,Y., Harada,N. et al. (2012) Engulfment of across cerebral endothelium. J. Immunol., 168, hematopoietic stem cells caused by down- 5832-5839. regulation of CD47 is critical in the pathogenesis of 58. Fukunaga,A., Nagai,H., Noguchi,T., Okazawa,H., hemophagocytic lymphohistiocytosis. Blood, 120, Matozaki,T., Yu,X., Lagenaur,C.F., Honma,N., 4058-4067. Ichihashi,M., Kasuga,M. et al. (2004) Src 69. Quang Van,V., Raymond,M., Baba,N., Rubio,M., homology 2 domain-containing protein tyrosine Wakahara,K., Susin,S.A. and Sarfati,M. (2012) phosphatase substrate 1 regulates the migration CD47(high) expression on CD4 effectors identifies of Langerhans cells from the epidermis to draining functional long-lived memory T cell progenitors. lymph nodes. J. Immunol., 172, 4091-4099. J. Immunol., 188, 4249-4255. 59. Li,L.X., Atif,S.M., Schmiel,S.E., Lee,S.J. and 70. Jaiswal,S., Jamieson,C.H., Pang,W.W., Park,C.Y., McSorley,S.J. (2012) Increased susceptibility to Chao,M.P., Majeti,R., Traver,D., Van Rooijen.N. Salmonella infection in signal regulatory protein and Weissman,I.L. (2009) CD47 is upregulated on alpha-deficient mice. J. Immunol., 189, 2537-2544. circulating hematopoietic stem cells and leukemia 60. van den Vijver,E., Maddalena,A., Sanal,O., cells to avoid phagocytosis. Cell, 138, 271-285. Holland,S.M., Uzel,G., Madkaikar,M., de Boer,M., 71. Subramanian,S., Boder,E.T. and Discher,D.E. van Leeuwen,K., Koker,M.Y., Parvaneh,N. et al. (2007) Phylogenetic divergence of CD47 (2012) Hematologically important mutations: interactions with human signal regulatory leukocyte adhesion deficiency (first update). protein alpha reveals locus of species specificity. Blood Cells Mol. Dis., 48, 53-61. Implications for the binding site. J. Biol. Chem., 61. Friedl,P. (2004) Prespecification and plasticity: 282, 1805-1818. shifting mechanisms of cell migration. Curr. Opin. 72. Tsai,R.K. and Discher,D.E. (2008) Inhibition Cell Biol., 16, 14-23. of “self” engulfment through deactivation of 62. Oldenborg,P.A., Zheleznyak,A., Fang,Y.F., myosin-II at the phagocytic synapse between Lagenaur,C.F., Gresham,H.D. and Lindberg,F.P. human cells. J. Cell Biol., 180, 989-1003. (2000) Role of CD47 as a marker of self on red 73. Chao,M.P., Alizadeh,A.A., Tang,C., blood cells. Science, 288, 2051-2054. Myklebust,J.H., Varghese,B., Gill,S., Jan,M., 63. Oldenborg,P.A., Gresham,H.D., Chen,Y., Izui,S. Cha,A.C., Chan,C.K., Tan,B.T. et al. (2010) Anti- and Lindberg,F.P. (2002) Lethal autoimmune CD47 antibody synergizes with rituximab to hemolytic anemia in CD47-deficient nonobese promote phagocytosis and eradicate non- diabetic (NOD) mice. Blood, 99, 3500-3504. Hodgkin lymphoma. Cell, 142, 699-713. 64. Yamao,T., Noguchi,T., Takeuchi,O., Nishiyama,U., 74. Chao,M.P., Tang,C., Pachynski,R.K., Chin,R., Morita,H., Hagiwara,T., Akahori,H., Kato,T., Majeti,R. and Weissman,I.L. (2011) Extranodal Inagaki,K., Okazawa,H. et al. (2002) Negative dissemination of non-Hodgkin lymphoma regulation of platelet clearance and of the requires CD47 and is inhibited by anti-CD47 macrophage phagocytic response by the antibody therapy. Blood, 118, 4890-4901. transmembrane glycoprotein SHPS-1. J. Biol. 75. Spaargaren,M. (2011) Lymphoma spread? Target Chem., 277, 39833-39839. CD47-SIRPalpha! Blood, 118, 4762-4764.
24 76. Zhao,X.W., Kuijpers,T.W. and van den Berg,T.K. 80. Okuzawa,C., Kaneko,Y., Murata,Y., Miyake,A., (2012) Is targeting of CD47-SIRPalpha enough for Saito,Y., Okajo,J., Tomizawa,T., Kaneko,Y., 1 treating hematopoietic malignancy? Blood, 119, Okazawa,H., Ohnishi,H. et al. (2008) Resistance to 4333-4334. collagen-induced arthritis in SHPS-1 mutant mice. 77. Zhao,X.W., van Beek,E.M., Schornagel,K., Van der Biochem. Biophys. Res. Commun., 371, 561-566. Maaden,H., Van Houdt,M., Otten,M.A., Finetti,P., 81. Dong,L.W., Kong,X.N., Yan,H.X., Yu,L.X., Chen,L., Van Egmond.M., Matozaki,T., Kraal,G. et al. (2011) Yang,W., Liu,Q., Huang,D.D., Wu,M.C. and CD47-signal regulatory protein-alpha (SIRPalpha) Wang,H.Y. (2008) Signal regulatory protein alpha interactions form a barrier for antibody-mediated negatively regulates both TLR3 and cytoplasmic tumor cell destruction. Proc. Natl. Acad. Sci. U. S. pathways in type I interferon induction. Mol. A, 108, 18342-18347. Immunol., 45, 3025-3035. 78. Sagawa,M., Shimizu,T., Fukushima,N., Kinoshita,Y., 82. Kong,X.N., Yan,H.X., Chen,L., Dong,L.W., Yang,W., Ohizumi,I., Uno,S., Kikuchi,Y., Ikeda,Y., Yamada- Liu,Q., Yu,L.X., Huang,D.D., Liu,S.Q., Liu,H. et al. Okabe,H. and Kizaki,M. (2011) A new disulfide- (2007) LPS-induced down-regulation of signal linked dimer of a single-chain antibody fragment regulatory protein {alpha} contributes to innate against human CD47 induces apoptosis in lymphoid immune activation in macrophages. J. Exp. Med., malignant cells via the hypoxia inducible factor- 204, 2719-2731. 1alpha pathway. Cancer Sci., 102, 1208-1215. 83. Baral,P. and Utaisincharoen,P. (2012) Involvement of 79. Tomizawa,T., Kaneko,Y., Kaneko,Y., Saito,Y., signal regulatory protein alpha, a negative regulator Ohnishi,H., Okajo,J., Okuzawa,C., Ishikawa- of Toll-like receptor signaling, in impairing the Sekigami,T., Murata,Y., Okazawa,H. et al. (2007) MyD88-independent pathway and intracellular Resistance to experimental autoimmune killing of Burkholderia pseudomallei-infected encephalomyelitis and impaired T cell priming mouse macrophages. Infect. Immun., 80, 4223-4231. by dendritic cells in Src homology 2 domain- containing protein tyrosine phosphatase substrate-1 mutant mice. J. Immunol., 179, 869-877.
25
SIRPα controls the activity of the phagocyte NADPH oxidase by restricting 2 the expression of gp91phox Summary The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host controlling its activity is essential. Little is known about the endogenous mechanism(s) that limit NADPH oxidase activity. Here we demonstrate that the myeloid inhibitory receptor SIRPα acts as a negative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRPα-mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRPα in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRPα and its natural ligand CD47 as well as signaling through the SIRPα cytoplasmic ITIM motifs. Suppression of the respiratory burst by SIRPα was caused by a selective repression of gp91phox expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRPα can limit gp91phox expression during myeloid development and thereby controls the magnitude of the respiratory burst in phagocytes.
Ellen M. van Beek*1, Julian Alvarez Zarate*1, Robin van Bruggen1, Karin Schornagel1, Anton Tool1, Takashi Matozaki2, Georg Kraal3, Dirk Roos1, Timo K. van den Berg1
1Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX, Amsterdam, The Netherlands 2Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan 3Department of Molecular Cell Biology and Immunology, VU Medical Center, 1081 BT, Amsterdam, The Netherlands
* these authors contributed equally to this work
Cell Rep. 2012 Oct 25;2(4):748-55
28 Introduction One of the most important anti-microbial activities of phagocytes is the abrupt formation of reactive oxygen species (ROS), a process known as the respiratory burst. This is mediated by the 2 phagocyte NADPH oxidase complex, and its importance is best illustrated by patients with chronic granulomatous disease (CGD) that have a dysfunctional NADPH oxidase and as a result are hyper- susceptible to a variety of bacterial and fungal infections(1;2). The phagocyte NADPH oxidase is a multi-subunit enzyme complex composed of: i) the membrane proteins, gp91phox (NOX2), the catalytic component of the oxidase, and p22phox, ii) the cytosolic proteins p40phox, p47phox, p67phox and iii) the small GTPase Rac. Activation of the oxidase involves translocation of the cytosolic subunits p40phox, p47phox, p67phox and Rac to the plasma membrane and assembly of the oxidase - complex. Once assembled, NADPH oxidase generates superoxide (O2 ) formation, by transferring electrons from NADPH in the cytosol over the plasmamembrane to molecular oxygen. Superoxide produced by the oxidase forms the basic compound from which other ROS, such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), are formed. High concentrations of ROS are directly toxic to microbes, and perhaps also indirectly by the liberation of hydrolytic proteases(3). The mechanisms of NADPH oxidase activation have been relatively well characterized, but essentially nothing is known on whether and how the magnitude of the respiratory burst is controlled. The latter is important since ROS do not only play a critical role in host defense, but can also be toxic to the host. The tight control over NADPH oxidase activity is illustrated amongst other things by the observation that there is only very little interindividual variation in the respiratory burst (Zhao et al, unpublished). The magnitude of the respiratory burst is likely to be primarily determined by the protein expression levels of the various NADPH oxidase components, which are expressed in a developmentally regulated fashion in phagocytes. While the developmental pathways and transcription factors that trigger the expression of the different NADPH oxidase components during myeloid development have been established (4-6), putative regulatory mechanisms that counterbalance these and that prevent excessive potentially harmful expression of the various NADPH oxidase components within phagocytes have remained unknown. SIRPα is a inhibitory immunoreceptor predominantly expressed on myeloid and neuronal cells (7;8). The cytoplasmic region of SIRPα contains four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which upon ligand binding become phosphorylated, and mediate the recruitment and activation of the SH2-domain-containing tyrosine phosphatases (PTPase) SHP-1 and SHP-2. SHP-1 and SHP-2 can in turn dephosphorylate specific protein substrates and thereby mediate various biological functions, generally in a negative fashion. The N-terminal V-like Ig domain mediates recognition of the broadly expressed transmembrane glycoprotein CD47 (9-14). SIRPα has been implicated in the regulation of a number of functions in myeloid cells (15;16). One of the best documented functions of SIRPα is its inhibitory role in the phagocytosis of host cells by macrophages. In particular, the ligation of SIRPα on macrophages by the ‘don’t eat me’ signal CD47 expressed on host cells, such as erythrocytes and platelets, generates an inhibitory signal that negatively regulates phagocytosis (17;18). Based on this is appears that CD47 acts as a molecular signature of ‘self’ that by interacting with the ‘self sensor’ SIRPα on phagocytes and
29 other myeloid cells limits immune-mediated damage against normal and healthy host cells during infection and inflammation. However, until now a direct involvement for CD47-SIRPα interactions in the regulation of inflammatory mediators and anti-microbial functions has not been reported. Here we demonstrate that SIRPα acts as a critical negative regulator of the respiratory burst. Inhibition of the phagocyte NADPH oxidase by SIRPα involves interactions between SIRPα and the ‘self’ molecule CD47 as well as signaling through the SIRPα ITIMs, which result in a selective suppression of gp91phox expression. This mechanism may help to prevent collateral oxidative damage to the host during infection and other inflammatory conditions.
Results and Discussion Enhanced NADPH oxidase activity in SIRPα-mutant phagocytes To investigate whether SIRPα signaling regulates the phagocyte NADPH oxidase we performed studies with cells from SIRPα-mutant mice. These mice express a SIRPα molecule lacking the cytoplasmic tail and signaling capacity (18). Distinct bone marrow cell populations of SIRPα- mutant and control mice, including granulocytes, monocytes, immature myeloid cells, and lymphoid cells, were FACS-sorted using CD31 and Ly-6C as markers (Fig.1A) as described previously (19), and their PMA-induced respiratory burst was analyzed. As reported before there are no detectable differences in bone-marrow composition between the mutant and control mice, essentially excluding a prominent non-redundant role of SIRPα signaling in myeloid differentiation(20). We observed a significantly (50-75%) enhanced respiratory burst activity in granulocytes and monocytes from SIRPα-mutant mice in comparison to cells from wild-type mice (Fig.1B). A similar difference was seen in bone marrow-derived macrophages (Fig.1C). The respiratory burst in immature myeloid cells (Fig.1B) appeared unaffected. The production of other inflammatory mediators, including nitric oxide, TNFα, IL1β, IL6 and IL10, by bone marrow-derived macrophages in response to LPS was not significantly affected when comparing wild-type and SIRPα-mutant mice (Fig.2). Collectively, these data indicate a selective inhibitory role for SIRPα signaling in the regulation of the respiratory burst. The lack of difference between WT and SIRPα-mutant phagocytes appears in contrast to previous reports (21;22), in which knock-down of SIRPα in macrophages was shown to enhance cytokine production in response to TLR ligands. One way to explain this apparent discrepancy is that inhibition of cytokine production can only be caused by a complete absence of SIRPα, but not by selective deletion of its cytoplasmic tail. A more trivial explanation could relate to differences in the method of interference. We have used macrophages from gene-targeted knock-out mice, whereas the other studies used shRNA- and siRNA-mediated knock-down, which could also have triggered macrophage danger pathways that may have contributed to the response(23). At least our current findings essentially exclude a regulatory role for SIRPα signaling in TLR-induced cytokine production and support the idea that SIRPα signaling is regulating selected inflammatory mediators, such as ROS.
30 A 1: early blasts 2: lymphoid cells 3: erythroid cells 4: myeloid cells (immature) 1 4 2 5: granulocytes CD31 2 6: monocytes 5 6 3
Ly-6C B C P=0.002 1000 SIRPalpha WT 90 P=0.002 900 SIRPalpha mutant
) 80 800 70 700 60 600 NS 50 500 40 400 30 300
200 20 Hydrogen peroxide (nM/min peroxide Hydrogen P=0.03 10
100 Hydrogen (nM/min) peroxide
0 0 control PMA monocytes monocytes granulocytes granulocytes myeloid cells myeloid cells myeloid lymphoid cells lymphoid cells lymphoid
control PMA
Figure 1. Phagocyte NADPH oxidase activity is enhanced in macrophages and granulocytes from SIRPα-mutant mice. (A) Flow cytometric double labeling of bone-marrow cells for CD31 and Ly-6C, identifying the major subpopulations of hematopoietic cells. Sorted populations of (B) monocytes, granulocytes, lymphocytes and immature myeloid cells, or (C) cultured bone marrow-derived macrophages from wild-type (white bars) or SIRPα-mutant (black bars) C57BL/6 mice were evaluated for PMA-induced NADPH-oxidase activity. Data are the means ± SD of five animals, with each measurement being performed in triplicate. Significance was determined by Student’s t-test. See also Figure 2.
SIRPα overexpression inhibits the NADPH oxidase in human phagocytic cells To investigate the mechanism by which SIRPα regulates the phagocyte respiratory burst we tested the effect of overexpression of SIRPα in human myeloid PLB-985 cells. PLB-985 cells are suitable for studying NADPH oxidase activity(24) and were found to express relatively low levels of endogenous SIRPα as shown by Western blotting (Fig.3A) and flow cytometry (Fig.4A). A chimeric rat-human SIRPα protein was expressed in PLB-985 cells because it would allow selective monitoring and manipulation by the agonistic mAb ED9, specifically directed against the rat SIRPα extracellular domain (8;25). PLB-985 cells, or mutants with a targeted mutation of the gp91phox gene (PLB-985 X-CGD) (24), were retrovirally transduced with full-length chimeric rat-human SIRPα protein (SIRPα-WT), or a SIRPα deletion mutant (SIRPα-Δ87) that is unable to
31 WT ABSIRP mutant 10 3000 n.s. n.s. 8 2000 6 g/ml) (pg/ml) 4 1000 NO ( TNF 2
0 0 S LP /IFN control S control LP CD 20 n.s. 1500 n.s.
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ol ol LPS LPS contr contr
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Figure 2. NO and Cytokine Production by SIRPα Mutant Macrophages. (A–E) Bone-marrow-derived macrophages from WT and SIRPα mutant macrophages were cultured in the presence of 100 ng/ml E. coli LPS, or a combination of 400 U/ml of IFNg and LPS. NO production and cytokines were measured as described in Materials and Methods at 4 h (TNFα) or 20 h (NO, IL1β, IL6, and IL10). Data are presented as the mean ± SD of triplicate measurements; n.s., nonsignificant. Note that there are no detectable differences between WT and SIRPa mutant mice.
signal because it lacks the cytoplasmic tail. Flow cytrometric analysis showed that the levels of SIRPα expression were comparable for the different cell lines generated (Fig.5A) and similar to those generally seen on rat myeloid cell lines or primary rat myeloid cells, such as macrophages or granulocytes (8) (not shown). Western blotting with an antibody against the cytoplasmic tail of SIRPα identified both endogenous and chimeric SIRPα proteins and confirmed that SIRPα-Δ87 cells express a truncated SIRPα (Fig.3A).
32 A PLB-985 PLB-985 X-CGD
SIRP (exogenous) 2
SIRP (endogenous)
actin 87 87 EV EV -WT -WT - - SIRP SIRP SIRP SIRP
B
C
Figure 3. Properties of PLB-985 Cell Lines Expressing WT and Δ87 Versions of Chimeric Rat-Human SIRPα. (A) SIRPα expression in PLB-985 cell lines. SIRPα protein expression in PLB cells was evaluated by western blotting with an antibody directed against the cytoplasmic tail of SIRPα. Differential glycosylation allows the distinction between the chimeric rat-human exogenous and human endogenous SIRPα, which carry 14 and three potential N-glycosylation sites in their extracellular regions, respectively. The lack of antibody reactivity with the exogenous molecule in the case of the SIRPα-Δ87 cell lines in combination with anti-rat SIRPα reactivity detected by flow cytometry (Figure 2A) confirms the absence of the cytoplasmic tail for the truncated SIRPα. β-actin was used as a loading control. (B and C) SIRPα is a negative regulator of both PMA- and STZ-induced respiratory bursts. NADPH oxidase activity in the indicated PLB-985 cell lines induced with buffer (white bars), PMA (black bars), or STZ (gray bars) after 5–6 days of either granulocytic (B) or monocytic (C) differentiation. Values shown represent the mean ± SD of three independent experiments, each performed in triplicate; *p < 0,05, **p < 0,001, by Student’s t test.
33 A
ratSIRP huSIRP huSIRP1 CD47
EV
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Y1234F
∆ECD
∆V
V56M
B 87 ECD V V56M EV WT ∆ ∆ ∆
-130 endogenous SIRP -95
-55
-34 -26
-17
Figure 4. Characterization of PLB-985 Cell Lines Expressing WT and Mutant Versions of Chimeric Rat-Human SIRPα. (A) Flow-cytometric analysis of the surface expression of the exogenous rat-human SIRPα (using anti-rat SIRPα mAb ED9), endogenous human SIRPα (with antihuman SIRPα/β mAb 7C2 and anti-human SIRPα1 mAb 1.23A), and CD47 (with mAb B6H12) on the indicated PLB-985 transfectants. All cell lines express comparable levels of exogenous and endogenous SIRPα molecules and CD47. Also note that staining with mAb ED9 is absent in the ΔECD and ΔV cells (but not V56M) cells, demonstrating that ED9 recognizes the N-terminal V-type of domain of rat SIRPα. (B) Expression of the SIRPα extracellular region mutants in PLB-985 cells evaluated by western blotting with an antibody against the SIRPα cytoplasmic domain. Differential glycosylation allows discrimination between the WT chimeric rat-human exogenous and human endogenous SIRPα.
34 The respiratory burst was studied in the different PLB-985 cells after in vitro granulocytic or monocytic differentiation using dimethylformamide (DMF) or vitamin D3 (VitD3), respectively. PMA-induced NADPH oxidase activity was normal in control cells, but was abolished in cells expressing wild type SIRPα (Fig.5B,C). This effect occurred after either granulocytic or monocytic 2 differentiation. Clearly, the inhibitory effect was not observed in the SIRPα-Δ87 mutant, suggesting that SIRPα signaling was required. In fact, the SIRPα-Δ87 cells generated a response that was considerably higher than that of the empty vector cells, suggesting that the mutant SIRPα protein acted as a dominant-negative protein, by competing e.g. with endogenous SIRPα for CD47 binding (see below). Importantly, all responses were entirely attributable to the gp91phox (NOX2)-containing phagocyte NADPH oxidase, as they were completely absent in the PLB-985 X-CGD cells, which have a targeted mutation of the gp91phox-encoding CYBB gene. Among several other stimuli of NADPH oxidase activation tested (i.e. serum-treated zymosan, fMLP and human IgG complexes) only serum-treated zymosan (STZ) generated a measurable response in the PLB-985 cells, and again this was completely abrogated by SIRPα-WT expression (Fig.3B). This is consistent with a generalized effect of SIRPα on the NADPH oxidase.
SIRPα selectively represses gp91phox expression during myeloid differentiation Activation of the multi-subunit NADPH oxidase complex requires assembly of its individual components, which are expressed during myeloid differentiation(1). To establish the basis for SIRPα-dependent regulation of the respiratory burst, we investigated the expression levels of the different components of the NADPH oxidase complex upon myeloid differentiation. Expression of the membrane component gp91phox, which forms the enzymatic core of the phagocyte NADPH oxidase, was evaluated in undifferentiated, and in granulocytic and monocytic PLB-985 cells by Western blotting. The differentiation-induced rise in gp91phox expression was completely absent in cells that express the full-length SIRPα protein (SIRPα- WT) (Fig.5D). The same was observed when surface levels of gp91phox were analyzed by flow cytometry with 7D5 mAb (Fig.6A). Also, the enhanced respiratory burst activity in cells that express the truncated receptor (SIRPα-Δ87) was associated with a higher gp91phox expression. Importantly, the levels of two of the other NADPH oxidase components, p67 and p47, remained unaffected by overexpression of full-length or truncated SIRPα (Fig.5D), suggesting that SIRPα was selectively regulating gp91phox expression. However, the similar levels of upregulation of p47 and p67 observed in the empty vector, SIRPα-WT and SIRPα-Δ87 cells also indicated that differentiation was unaffected by SIRPα-WT or SIRPα-Δ87 introduction, suggesting that SIRPα was not regulating differentiation in general. Furthermore, SIRPα did not affect the upregulation of other myeloid differentiation markers, such as CD11b and CD14, during granulocytic or monocytic differentiation (not shown). The upregulation of endogenous SIRPα on PLB-985 cells coincided with that of gp91phox around day 1-3 of neutrophilic differentiation (Fig.6B). To demonstrate that gp91phox was indeed the only relevant factor down-regulated by SIRPα, gp91phox was reconstituted by retroviral expression into SIRPα-WT cells (Fig.5E) and this resulted in a full restoration of the respiratory burst (Fig.5F). A similar restoration was observed when
35 A PLB-985 PLB-985 X-CGD D undifferentiated DMF VitD3
EV
- gp91phox
SIRP-WT
- p67phox - p47phox
SIRP-87 EV EV EV 87 87 87 -WT -WT -WT - - - SIRP SIRP SIRP SIRP SIRP SIRP SIRP
B E control gp91phox
PLB-985 PLB-985 X-CGD X-CGD
SIRP-WT
F C
PLB-985 PLB-985 X-CGD
Figure 5. SIRPα overexpression in PLB-985 cells inhibits the respiratory burst by repressing gp91phox expression. PLB-985 and gp91phox PLB-985 X-CGD cells were stably transduced with empty vector, chimeric rat-human SIRPα (SIRPα-WT) or an N-terminal truncated SIRPα protein lacking 87 amino acids of the cytoplasmic tail of SIRPα (SIRPα-Δ87). (A) SIRPα surface expression was evaluated by flow cytometry with Alexa 633-conjugated ED9 directed against rat SIRPα antibody (filled histograms). Unstained cells are indicated as control (open histogram). (B,C) PMA-induced NADPH-oxidase activity in the indicated PLB-985 (white bars) and PLB-985 X-CGD (black bars) cell lines after 5-6 days of either granulocytic (B) or monocytic (C) differentiation induced with DMF of vitamin D3, respectively. Values shown in panels B and C represent means ± SD of 15 measurements from 5 independent experiments. *, p < 0,001, by Student’s t test. (D) Expression levels of gp91phox, p67phox and p47phox determined by Western blotting in undifferentiated PLB-985 cells or those differentiated with DMF or vitamin D3 into granulocytic or monocytic cells, respectively. Note the lack of gp91phox, but not that of p67phox or p47phox, in SIRPα-WT cells. (E) Restoration of gp91phox expression after reconstitution of gp91phox in SIRPα-WT and X-CGD empty vector cells by retroviral transduction. The expression of gp91phox before (left panel) and after (right panel) retroviral transduction was evaluated by flow cytrometry after incubation with mAb 7D5 and goat-anti-mouse- IgG1 Alexa 633 antibody (solid histogram) or stained with isotype-matched antibody (open histogram). (F) PMA- induced NADPH-oxidase activity in granulocytic SIRPα-WT and X-CGD empty vector cells (white bars) in which gp91phox was reconstituted (black bars). Data are presented as the mean ± SD of three independent experiments each performed in triplicate. *, p < 0,001, by Student’s t test. See also Figures 3 and 6.
36 such reconstitution was performed in PLB-985 X-CGD cells. This shows that SIRPα suppresses phagocyte NADPH oxidase activity by a selective repression of gp91phox protein expression during myeloid differentiation. 2 Inhibition of the NADPH oxidase involves signaling via the SIRPα cytoplasmic ITIM motifs The experiments described above suggested that direct signaling through the cytoplasmic tail is involved in the regulation of the NADPH oxidase and gp91phox protein expression. The SIRPα cytoplasmic tail harbours ITIM motifs responsible for the recruitment of the cytosolic tyrosine phosphatases SHP-1 and SHP-2 (Fujioka et al. 1996; Kharitonenkov et al. 1997). Studies with dominant- negative SHP-1 in myeloid cells (26) and with phagocytes from motheaten SHP-1-deficient mice(27) demonstrated that at least SHP-1 acts as a negative regulator of the respiratory burst in myeloid cells. To investigate whether SHP-1 and/or SHP-2 recruitment by SIRPα plays a critical role in the suppression of the respiratory burst by SIRPα, each of the 4 tyrosines from the SIRPα ITIMs, or combinations thereof, were mutated into phenylalanines, and the resultant proteins were expressed in PLB-985 cells. To evaluate the binding of SHP-1 and SHP-2 to the SIRPα mutants we performed immunoprecipitation experiments. Analysis of the precipitates by Western blotting demonstrated constitutive binding of SHP-1 and SHP-2 to SIRPα and this was absent, or at least strongly reduced, by mutation of the ITIM tyrosines (Fig.7A). The same was observed in the reverse experiment i.e. SHP-1 or SHP-2 immunoprecipitation followed by Western blotting with SIRPα specific antibody (not shown). Mutation of all 4 ITIM tyrosines (SIRPα-Y1,2,3,4F) completely restored the respiratory burst (Fig.7B) and gp91phox protein expression (Fig.7C) to levels seen with the SIRPα-Δ87 cytoplasmic deletion mutant, suggesting that the ITIMs were responsible for the inhibitory activity. The level of inhibition obtained with the individual mutants correlated very well with their capacity to recruit SHP-1 and SHP-2. For instance, the membrane proximal Y1 appeared more important for the negative regulation of the NADPH oxidase than its membrane distal counterpart Y3. To obtain insight into the level of gp91phox regulation by SIRPα, we analyzed its mRNA levels by qPCR in the various PLB-985 mutants. Similar to the gp91phox protein expression, mRNA levels were low in the cells that express the full-length SIRPα protein (Fig.7D), suggesting suppression of transcriptional activity of the gp91phox/CYBB gene and/or increased mRNA turnover. There was a strong correlation between gp91phox mRNA, protein and NADPH oxidase activity. Finally, to investigate whether SIRPα expression and signaling also affected the intracellular microbial killing, the various PLB-985 mutants were evaluated for their capacity to kill intracellular Salmonella bacteria. Enhanced Salmonella outgrowth was observed in the PLB-985 X-CGD cells (Fig.6C) directly implicating a role for the NADPH oxidase in Salmonella killing. There was a good inverse relation between NADPH oxidase activity and intracellular bacterial survival (Fig. 7E). For instance, the overexpression of SIRPα-WT tended to enhance bacterial survival, whereas the SIRPα-Δ87 cytoplasmic deletion mutant resulted in bacterial survival lower than that observed in the empty vector cells. Also, the various Y-mutants displayed a pattern of microbial survival generally corresponding to their NADPH oxidase capacity. The various mutants displayed similar levels of Salmonella uptake (Fig.6D)
37 38 the Regulation of Endogenous SIRP quantified quantified relative to lamin B (Bii), and for SIRP bacteria for the andindicated binding periods, and phagocytosis werecytometry. quantified by flow cells expressing SIRP three measurements; *p < 0,05, by Student’s (D) t test. Salmonella binding and phagocytosis determined by PLB-985 of intracellularthe numbers bacteria were determined 24 h after challenge. Data are presented ± as SD the of mean oxidase. The PLB-985 and PLB-985 X-CGD cells were to allowed ingest S. enterica serovar Typhimurium 14028s, and Figure 6. SIRPa withcoincides that of gp91 and the expression of SIRP cells were differentiated into granulocytic cells in the presence MaterialsMethods, in and of DMF as described or monocytic cells, respectively.or monocytic gp91 Surface expression. The indicated PLB-985 were cell lines differentiated with for either6 days DMF or VitD3 into granulocytic Coordinated regulation of SIRP Regulation of gp91phox Expression and Phagocytosis Surface by SIRP and Salmonella Survival Bi A C
Salmonella survival SIRP -87 SIRP -WT empty vector 10000 12500 2500 5000 7500 nifrnitdDMF undifferentiated 0135days 0 α
mutants. The indicated PLB-985 were cell lines incubated with FITC-labeled Salmonella con t rol 16,8% 23,9% 1,8% α X
-
and gp91 C GD α and gp91 phox . (C) Salmonella killing by PLB-985 cells is dependent on the phagocyte NADPH gp91 D α laminB - gp91 - SIRP - during Myeloid Differentiation. during Myeloid (A) SIRP phox phox 35,6% 58,6% phox 6,9% 100 was determined by western blotting (Bi). The fluorescent signal was Salmonella phagocytosis (%)
e 20 40 60 80 m p 0
expression during granulocytic differentiation cells. of PLB985 PLB985 ty
ve phox
phox cto α r 1 by flow cytometry (Biii). Note that the upregulation cytometry 1 by flow of (surface) levels were evaluated by flow cytometry with the 7D5 mAb. were levels evaluated cytometry (B) by flow
wild type Bii
RFU vitD3 0 1 2 3 4 d 47,9%
a 21,2% y 5,2% d87 0 d SIRP a y 1 day 3
Y 1 d F a y 5
Y3F RFU 0 1 2 3 4 d a y gp91phox Y 1 0 ,2 day 1 F da y Y 3 3 da ,4 F y 5 α suppresses gp91phox surface Y2,4F Biii MFI 2000 4000 6000 8000 Y1 SIRP ,2 0 ,3 d ,4 ay F 0 d a y 1 d a y 3 30 min 30 min 10 d 1 a y 5 α , and A
2
600 B undifferentiated
) 500 DMF 400 vitamin D3 300 200
100 (nM/min peroxide Hydrogen 0 empty vector wild type d87 Y1F Y3F Y1,2F Y3,4F Y2,4F Y1,2,3,4F 70 C 60 50 40 30 20 10 gp91phox positive cells (%) 0 empty w ild type d87 Y1F Y3F Y1,2F Y3,4F Y2,4F Y1,2,3,4F D 1 vector
0,8 0,6 0,4 0,2
gp91phox mRNA(relative abundance) 0 F F F F F V T 3 F 30000 B-E -d87 Y1,2 aY3,4 E PL PLB-W PLB IRPa IRP B-SIRPaY PLB-SIRPaY1 PL 25000 PLB-S PLB-S PLB-SIRPaY2,4 l PLB-SIRPaY1,2,3,4 20000 15000
10000 Salmonella surviva 5000 0 empty w ild type d87 Y1FF Y3FF Y1,2F Y3,4F Y2,4F Y1,2,3,4F 87 4F vector WT ∆ Y1 Y3 ,2F ,4F - - - - -Y1 -Y3 -Y2, P P P R R I R P P -Y1,2,3,4F SI S SI SIRP IR IR IRP Empty vector S S S SIRP
Figure 7. The cytoplasmic ITIMs of SIRPα are required for the inhibition of the NADPH oxidase. (A) SHP-1 and SHP-2 binding to SIRPα-WT, SIRPα-Δ87 and SIRPα tyrosine mutants evaluated by immunoprecipitation of SIRPα, with mAb ED9 against rat SIRPα, and Western blotting with antibodies against SHP-1, SHP-2 and SIRPα. (B) PMA-induced NADPH-oxidase activity in differentiated granulocytic or monocytic PLB-985. Data are presented as the mean ± SD of three independent experiments each performed in triplicate. (C) Expression of surface gp91phox in differentiated granulocytic, monocytic or undifferentiated PLB-985 cells analyzed by flow cytometry uing 7D5 mAb. Data are presented as the mean ± SD of three independent experiments. (D) mRNA level of gp91phox in granulocytic, monocytic or undifferentiated cells detected by quantitative RT-PCR. Data are presented as the mean ± SD of two independent experiments each performed in duplicate. (E) Intracellular killing of Salmonella bacteria. The different cell lines were allowed to ingest S. enterica serovar Typhimurium 14028s and the numbers of intracellular bacteria were determined at 24h after challenge. The level of Salmonella uptake at the start of the experiment were comparable for all cells (not shown). Data are presented as the mean ± SD of three independent experiments performed in triplicate. *, p < 0,05, by Student’s t test between the indicated conditions and the empty vector control. See also Figure 6.
39 Collectively, these results show that SIRPα signaling via the ITIMs negatively regulates the respiratory burst by controlling the expression of gp91phox. It should be noted that we have not yet been able to characterize the relevant downstream signaling pathway(s). Of note, two transcription factor complexes, ICSBP and HoxA10, have previously been shown to play a key role in the regulation of gp91phox gene expression, and both of these are also subject to regulation by SHP-1 and/or SHP-2(4-6). Both complexes constitute obvious candidates to mediate the effects of SIRPα signaling on gp91phox gene expression, but our analysis of their activity by electrophoretic mobility shift assay in the cell panel studied here did not provide any evidence for their involvement (JAZ, unpublished). We are currently exploring alternative possibilities.
Inhibition of the NADPH oxidase by SIRPα involves CD47-SIRPα interactions SIRPα has been shown to interact via its N-terminal Ig-like domain with the broadly expressed CD47 molecule, and the molecular basis for CD47-SIRPα interactions has been established by mutagenesis and crystallography (12;14). SIRPα ligation by CD47 triggers SIRPα ITIM phosphorylation, SHP-1 and/or SHP-2 recruitment and signaling, and this regulates downstream cellular responses. As indicated above, the observation that SIRPα mutants, such as the SIRPα-Δ87 and several of the ITIM tyrosine mutants, display an enhanced oxidase activity and gp91phox expression suggested a dominant-negative effect. We anticipated that the rat extracellular domain of the chimeric SIRPα molecule that was introduced into the PLB-985 cells would compete with the endogenous human SIRPα for CD47 binding, thereby reducing inhibitory signaling through the latter. Indeed, PLB-985 cells express CD47 on their surface (Fig.8A) and CD47 expression levels were not significantly affected by expression of SIRPα or its mutants (Fig.4A). To directly address whether CD47-SIRPα interactions contribute to the inhibitory effect of SIRPα on the phagocyte respiratory burst, several SIRPα variants were constructed in which the extracellular ligand-binding domain was mutated. The mutants included deletions of the entire extracellular region (SIRPα-ΔECD), the N-terminal V-like Ig domain (SIRPα-ΔV), or a single point mutation V56M within the N-terminal V-like Ig region that was previously demonstrated to abolish CD47 binding (12). The resulting cell lines were analyzed by FACS with antibodies against rat and human SIRPα (Fig.4A) and Western blotting (Fig.4B) and were found to express comparable surface levels of the various SIRPα molecules, with the expected sizes. In all of the cells with SIRPα extracellular domain mutations the respiratory burst was strongly enhanced, as compared to empty vector controls, yielding activities close to those of the Δ87 mutant (Fig.8B). This suggested that similar to the mutants that affected SIRPα signaling, also the mutants that affect ligand binding were acting as dominant negative molecules, the latter likewise by sequestering relevant downstream signaling molecules, such as SHP-1 and/or SHP-2. It was clearly important to demonstrate a direct interaction between the rat extracellular domains of our chimeric SIRPα constructs and human CD47. For this purpose, we generated a fusion protein of the extracellular domain of human CD47 and the Fc part of IgG1 (CD47-Fc) and developed a fluorescent bead assay to measure cellular CD47 binding. Analysis of the mutants demonstrated enhanced CD47 binding in SIRPα-WT cells as compared to empty vector cells
40 (Fig.8C). This enhanced binding was prevented by blocking with the anti-rat SIRPα-specific mAb ED9, directly demonstrating that the chimeric rat-human SIRPα molecules are capable of binding human CD47. In addition, this analysis also demonstrated detectable CD47 binding by the endogenous human SIRPα that could be inhibited by the mAb 7C2. Analysis of the other 2 mutants confirmed CD47 binding to SIRPα-WT, SIRPα-∆87 and SIRPα-Y1,2,3,4 cells, but not to any of the SIRPα extracellular domain mutants (Fig.8D). Collectively, these results indicate that CD47-SIRPα interactions contribute to the inhibitory activity of SIRPα on the respiratory burst. Clearly, we are formally unable to distinguish whether cis- (i.e. on the same cell) and/or in trans- (i.e. between different cells) interactions
A B 300 DMF ) 250 vitamin D3 200 150 100
Hydrogen peroxide Hydrogen (nM/min 50 CD47 0 T V orctor 87 -∆ 6M --WWT ∆ ECD 5 e - vect Pa -∆ -V56M-V y R a pt RP IRP SI IRP S IRPP em SI S SIR empty v SIRP S
C D 18000 ED9 7C2 ED9 + 7C2 ED9-blockable 16000 7C2-blockable 14000 remaining EV 12000
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4000 WT bead bindingCD47 (MFI) 2000
0 V 87 ∆ EV WT ∆ ECD V56M ∆ Y1234F
Figure 8. CD47-SIRPα interactions are instrumental in the suppression of the respiratory burst by SIRPα. (A) CD47 expression on PLB-985 cells as demonstrated by flow cytometry with mAb B6H12. (B) PMA-induced NADPH-oxidase activity in granulocytic (black bars) or monocytic (grey bars) PLB cells expressing each of the SIRPα extracellular mutants (SIRPα-ΔECD, SIRPα-ΔV or SIRPα-V56M. Data are presented as the mean ± SD of three independent experiments each performed in triplicate. *, p<0,05, by Student’s t test between the indicated conditions and the empty vector control. (C) Binding of fluorescent beads coated with human CD47-Fc protein to PLB-985 cells expressing rat-human chimeric SIRPα (WT) or empty vector (EV) cells. Histograms show the total CD47-bead binding (white area) and CD47-bead binding after preincubation of the cells with blocking mAb anti-rat SIRPα (ED9), anti-human SIRPα (7C2), or both (ED9 + 7C2) (grey area). (D) CD47-bead binding to the indicated mutants. The bars show total binding in the absence of antibodies as well as the effects of preincubation with anti-rat SIRPα mAb ED9 alone (ED9-blockable), the calculated difference between preincubation with both mAb ED9 plus the anti-human mAb 7C2 and preincubation with ED9 (7C2-blockable), and preincubation with both ED9 and 7C2 mAb (remaining). See also Figure 4.
41 are involved. However, considering the relatively low culture density of our cells and the low number of interactions between cells that occur during culture, we think it is most likely that the observed effects occur primarily as a result ofcis interactions. However, it would seem that in the context of a hematopoietic tissue such as the bone marrow in vivo where myeloid cells develop normally the propensity of trans interactions would be much higher and these may be also contribute in restricting gp91phox expression. Taken together these results demonstrate that CD47-SIRPα interactions and ITIM-dependent downstream signaling via SIRPα control the magnitude of the phagocyte respiratory burst by regulating the expression levels of gp91phox. We propose that CD47-SIRPα interactions participate in a homeostatic pathway acting on developing phagocytes that functions to control excessive NADPH oxidase activity, and this is anticipated to serve in the protection of host cells and tissues against ‘collateral’ oxidative damage during infection and other inflammatory conditions. Among the next challenges will be to provide insight into the mechanism(s) by which SIRPα- signaling regulates gp91phox expression, and to establish a contribution of CD47-SIRPα-dependent regulation of the NADPH oxidase during inflammation and infection. The latter question may perhaps not be straightforward to answer, because other relevant processes, such as e.g. leukocyte transendothelial migration, might also be regulated by CD47 and SIRPα as well (28;29).
Experimental procedures Antibodies The following mAb were used in this study: ED9, mouse IgG1 directed against rat SIRPα (unconjugated or Alexa 633-labelled); 7C2, mouse IgG1 directed against human SIRPα and β, which blocks interactions between SIRPα and CD47 (Santa Cruz, CA, USA); 1.23A, mouse IgG1 phox directed against human SIRPα1(30); 7D5, mouse IgG1 directed against human gp91 , a kind gift of dr. M. Nakamura (Nakasaki, Japan); B6H12, mouse IgG1 against human CD47 (kindly provided by dr. E. Brown (San Fransisco, CA, USA); ED2, mouse IgG1 against rat CD163 (isotype control); FITC- conjugated ER-MP20, rat IgG2a against Ly-6C, and biotinylated ER-MP12, rat IgG2a against CD31, were generously provided by dr. P. Leenen (Rotterdam, The Netherlands). Rabbit polyclonal Ab8120 (Abcam, Cambridge, United Kingdom) is directed against the cytoplasmic tail of human SIRPα. SH-PTP1, rabbit polyclonal antibodies against SHP-1 and lamin B, mouse monoclonal IgG1 directed against SHP-2, were from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit polyclonal antisera raised against recombinant human p47phox and p67phox were kindly provided by Dr. William Nauseef, (Iowa City, IA, USA). Alexa 633 goat-anti-mouse IgG (Molecular Probes, Eugene, OR, USA) and Alexa 633 streptavidin conjugate (Molecular Probes) were used as conjugates.
Mice and isolation of cell populations C57BL/6 mice with a targeted deletion of the SIRPα cytoplasmic region have been described previously (18). The mice that were originally generated onto the 129/Sv background had been backcrossed onto the C57BL/6 mice for ten generations. Wild-type C57BL/6 mice of the same genetic background were maintained together with the SIRPα-mutant mice in the breeding facility of the VU Medical Center,
42 Amsterdam. All mice were specified pathogen free. Permission for animal experiments described here was obtained from the Animal Welfare committee of the Vrije Universiteit in Amsterdam. Six- to eight-week-old SIRPα-mutant and wild-type mice were killed with a lethal intraperitoneal injection of Euthesate (8 mg sodium pentobarbital per mouse; Sanofi Santé 2 Animale Benelux B.V., Maassluis, The Netherlands). Tibiae were removed and cleaned of soft tissue and ground in a mortar with culture medium [α-Minimal Essential Medium (Gibco) supplemented with 5% (v/v) FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ ml amphotericin b (Antibiotic antimyotic solution, Sigma-Aldrich) and heparin (170 IE/ml; Leo Pharmaceutical Products B.V., Weesp, The Netherlands) as described (20). The cell suspension was aspirated through a 21-gauge needle and filtered over a 100-μm pore size Cell Strainer filter (Falcon/Becton Dickinson, Franklin Lakes, NJ, USA). Cells were washed twice in culture medium. For FACS sorting of distinct bone marrow cell populations, including granulocytes, monocytes, myeloid cells and lymphoid cells, bone marrow cells were labeled with ER-MP12bio and ER-MP20FITC. After 1 hour incubation at 4°C, cells were washed three times with PBS-0,1% (v/v) BSA. ER-MP12 binding was detected with Alexa 633-streptavidin conjugate (Molecular Probes) for 30 min at 4°C. After three washes with PBS-0,1% (v/v) BSA, cells were analyzed and FACS sorted (FACS Star Plus Becton Dickinson), and subsequently used to measure the respiratory burst activity as described below. Purity of the cell populations was checked by May-Grünwald/Giemsa staining and was comparable to that described previously(31). To obtain bone marrow-derived macrophages the freshly isolated bone-marrow cells were prepared as described above and were resuspended in RPMI-1640 containing 15% (v/v) L929-cell conditioned medium (as a source of CSF-1), 10% (v/v) fetal bovine serum, 25 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. The cell suspension from one mouse was distributed evenly over two bacterial plastic Petri dishes. The plates were incubated at
37°C in 5% CO2. Every 3 days medium was removed and replaced with fresh medium. After seven days of culturing cells were used to measure the respiratory burst activity as described above. Where indicated mouse bone-marrow-derived macrophages were cultured O/N at 0,3x106/ml in complete medium containing 100 ng/ml LPS from E.coli (Sigma) and/or 400 U/ml IFNγ. PLB-985 and PLB-985 X-CGD cells (24), kindly provided by Dr. M. Dinauer (Indianapolis, IN, USA), were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM
L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (complete medium) at 37°C and 5% CO2. For granulocytic differentiation, the cells were exposed to 0,65% (v/v) dimethylformamide (Sigma- Aldrich, St Louis, MO, USA) for 5 to 6 days. Monocytic differentiation was achieved by culturing in the presence of 100 nM vitamin D3 (1alpha, 25-dihydroxyvitamin D3, Sigma-Aldrich) for 5 to 6 days.
Measurement of respiratory burst activity Activity of the respiratory burst after phorbol myristate acetate (PMA, Sigma-Aldrich) or serum-treated zymosan (STZ; ICN Biochemicals, Cleveland, OH, USA) stimulation in transduced PLB-985 or PLB-985 X-CGD cells was measured with the Amplex Red (10-acetyl- 3,7-dihydroxyphenoxazine) Hydrogen Peroxide Assay kit (Molecular Probes, Eugene, OR, USA) as described (32). For information on antibodies, mice, isolation and culture of other cells,
43 retroviral transductions, flow cytometric analysis, quantitative PCR, immunoprecipitation and Western blotting, and intracellular killing of Salmonella see extended experimental methods.
Measurement of inflammatory mediators Culture supernatants were harvested and NO production was measured with the Giess assay as described before (25). Cytokines were measured by ELISA (R&D systems) according to instructions by the manufacturer.
Retroviral vectors and transductions A rat-human SIRPα (SIRPα-WT) fusion construct was generated from cDNA and PCR fragments as follows: nt 1-1236 (count starts at first methionine) of the rat SIRPα cDNA(8) was fused to nt 1230-1509 of the human cDNA (prot. accession No NM_080792). The chimeric SIRPα protein contains amino acids 1-412 of rat and amino acids 411-503 of human SIRPα, resulting in a total length of 505 amino acids, including the signal sequence. The deletion construct SIRPα-delta87 (SIRPα-Δ87) was constructed by PCR from the rat-human fusion cDNA (Table 1). The amplified fragment was cloned into pcDNA3 (Invitrogen, Breda, The Netherlands) downstream of the BamH1-XhoI of the rat SIRPα cDNA. The SIRPα tyrosine mutants were generated by PCR mutagenesis (Quikchange Site-Directed Mutagenesis Kit, Stratagene, La Jolla, CA, USA) (Table 1). The tyrosine → phenylalanine point mutations were introduced at positions Y430, Y454, Y471 and Y497 (representing the tyrosines of the four ITIMs of SIRPα and here termed Y1,2,3 and 4, respectively) of the human SIRPα-WT protein. The extracellular mutants, SIRPα-ΔV and SIRPα-ΔECD, were generated by the introduction of a ApaI restriction site at aa 26 and 156 for the ΔV mutant and a XhoI restriction site at aa 26 and 351 for the ΔECD mutant. These restriction sites were generated by PCR mutagenesis. In the SIRPα extracellular mutant, SIRPα-V56M, a valine → methionine point mutation at position aa 56 was generated. The sequence of all constructs was confirmed by DNA sequencing. For retroviral transduction, SIRPα-WT, the deletion mutant SIRPα-Δ87, the SIRPα extracellular domain mutants and SIRPα tyrosine mutants were cloned into the retroviral expression vector pLZRSpMBN-linker-IRES-EGFP(NotI-)(33), which was obtained from Dr. H Spits (Amsterdam, The Netherlands). The phoenix-A packing cell line(34), provided by Dr. G. Nolan (Stanford University, Stanford, CA, USA) was transfected, using calcium phosphate, with one of the different retroviral construct containing the SIRPα-WT, SIRPα-Δ87, SIRPα extracellular domain mutants, SIRPα tyrosine mutants or control construct containing no SIRPα insert. After selection of transfected cells with puromycin (1 µg/ml) (Sigma-Aldrich), virus was harvested as previously described (33) and used for retroviral transduction of PLB-985 and PLB-985 X-CGD cells. The transduced eGFP-positive cells were subsequently selected by FACS sorting (FACS Star Plus, Becton Dickinson). Cell surface expression of SIRPα was determined by FACS analysis with ED9 mAb as described below. FACS-sorted PLB-985 and PLB-985 X-CGD cells expressing eGFP only (termed empty vector) were used as a control in subsequent experiments. Expression of SIRPα and/or EGFP was tested regularly (i.e. at least once a month), and in the experiments described the proportion of cells expressing SIRPα exceeded 90%.
44 The retroviral expression vector LZRS-IRES-GFP with gp91phox was generated in our laboratory. PLB-985-SIRPα-WT and PLB-985 X-CGD empty vector cells were retrovirally transduced with this construct as described above. Cell surface expression of gp91phox was determined by flow cytometric analysis with 7D5 mAb as described below. 2
Flow cytometric analysis For flow cytometric analysis, cells of the PLB cell line panel were resuspended in PBS-0,1% BSA and stained with saturating concentrations of ED9-Alexa 633, 7D5, 7C2, 1.23A and/or B6H12-PE mAb. After 1 hour incubation at 4°C, cells were washed three times with PBS-0,1%BSA. 7D5, 7C2 or 1.23A binding was detected with goat-anti-mouse-IgG Alexa 633, for 30 min at 4°C. After three washes with PBS-0,1%BSA, cells were analyzed on a FACS Calibur (BD Biosciences, San Jose, CA, USA). For the CD47-beads generation a fusion protein containing the extracellular domain of CD47 and a human Fc tail (CD47-Fc) was generated. The extracellular domain of human CD47(35) was amplified using the forward 5’-AGA TCG ATA TCC CAG CTA CTA TTT AAT AAA ACA AAA TC-3’ and reverse 5’-GAG ATC AGA TCT AAA CCA TGA AAC AAC ACG ATA TTT TAG-3’ and subcloned using EcoRV and Bgl-II into the pFUSE-hIgG1-Fc2 vector. For expression of the fusion protein Freestyle HEK cells (Invitrogen, Breda, The Netherlands) were co-transfected with the plasmid and p21, p27 and pSVLT. After 6 days the supernatant was collected and the CD47-Fc protein was purified on a protein G-sepharose beads column (Thermo Scientific, Breda, The Netherlands). The human CD47-Fc fusion protein was bound to streptavidin-coupled fluorescent beads. For the coupling to the beads, 20 µl goat-anti human Fc-biotin (0,5 mg/ml; Jackson Immuno Research Code 109- 066-098) was added to a mix of 15 µl of streptavidin-coupled beads (6,7x106 beads/µl) and 350 µl PBS with 0,5% BSA and 0,02% azide (PBA). This was incubated for 2 hours at 37°C under shaking after which the beads were centrifuged 20.000g at 4°C for 2 minutes and washed twice with 500 µl PBA and resuspended in 100µl PBA. 0,5 µg of the purified CD47-Fc protein in 300 µl PBA was added and this was incubated rotating overnight at 4°C. The beads with the coupled fusion protein were centrifuged and washed twice and resuspended in 100 µl PBA and stored at 4°C for a maximum of 6 months. For the beads binding assay 5x104 cells were incubated with 2 µl CD47-beads in 40 µl PBS/0,1%BSA at 370C for 45 min. Cells and beads were then washed once and measured by FACS.
Quantitative RT-PCR mRNA was isolated from monocytic, granulocytic or undifferentiated PLB-985 cells with an RNA isolation kit (Qiagen, Hilden, Germany). cDNA was prepared with the Superscript III first-strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer’s instruction. Primers used for gp91phox (CYBB gene) expression analysis were 5’-TGC AGA TCT GCT GCA ACT GCT GGA G-3’ and 5’-CAA AGT CTT TTG TTT CAG GCC TGT C-3’. Primers 5’-AAA TAT GTG GTT GGA GAG CTC ATT-3’ and 5’-CCGAGTGAAGATCCCCTTTTTA-3’ were used for β-glucuronidase (GUS), as a control. Amplification by PCR was performed on a LightCycler instrument (Roche, Almere, The Netherlands). All primer pairs used in qPCR amplified a single band with expected molecular weight as analyzed by agarose gel electrophoresis. The gp91phox (CYBB gene) band was sequenced and showed 100% homology to the published human CYBB sequence (Genebank acc. No NM_000397).
45 Immunoprecipitation and Western blot analysis For immunoprecipitation of SIRPα, 1,5x106 PLB-985 cells were washed twice in PBS and centrifuged,
and the cell pellet was lysed with Igepal lysis buffer (Sigma-Aldrich) containing 1 mM Na3VO4 and protease inhibitor cocktail (Roche) for 1 hour at 4ºC. The lysates were clarified by centrifugation and precleared by incubation with protein A-Sepharose beads (Amersham Biosciences, Uppsala, Sweden) for 1 hour. SIRPα was immunoprecipitated by addition of anti-SIRPα (ED9 mAb) bound to protein A beads for 1 hour at 4ºC. SHP-1 or SHP-2 were immunoprecipitated by addition of anti-SHP-1/2 (Santa Cruz Biotechnology). Beads were washed with Igepal lysis buffer containing
1 mM Na3VO4 and protease inhibitor cocktail and resuspended in SDS sample buffer. Western blot analysis was performed with anti-SHP-1/2 (Santa Cruz Biotechnology) and anti-SIRPα antibodies. The cellular expression level of gp91phox, p67phox and p47phox in granulocytic, monocytic or undifferentiated PLB-985 cells was determined by Western blot with mAb 48 (anti-gp91phox) and rabbit polyclonal antisera raised against p47phox and p67phox. Appropriate HRP- or IRDye®- conjugated secondary antibodies were used to visualize the proteins and the signal was detected with respectively Supersignal West Dura (Pierce, Rockford, IL, USA) and a Gelimager (Epi Chemi II Darkroom combined with a 12-bit SensiCam charged-coupled device camera driven by Labworks 4.0 (UVP, Inc., Upland, CA, USA), or by Odyssey (Li-Cor Biosciences).
Intracellular killing of Salmonella Single colonies of Salmonella enterica serovar Typhimurium 14028s, obtained from Dr. A.M. van der Sar (Vrije Universiteit, Amsterdam, The Netherlands), were grown overnight in Luria-Bertani (LB) medium at 37ºC while being shaken (225 rpm). The CFU in the inoculum were determined by plating serial dilutions. PLB cell line cells were exposed to 0,65% DMF for granulocytic differentiation as described above. After 6 days, cells were washed with RPMI-1640 containing 10% (v/v) FCS and 1 mM glutamine, and cells (2*105) were then incubated with S. enterica serovar Typhimurium 14028s at a 10:1 concentration of multiplicity of infection in Falcon round-bottom
tubes (Becton Dickinson, Meylan Cedex, France) while rotating for 30 min at 37ºC, 5% CO2. The cells were then washed with PBS and incubated for 1 h in medium supplemented with 100 µg of gentamicin/ml to kill extracellular bacteria, and washed again. This time point is designated time zero. Medium supplemented with 10 µg of gentamicin/ml was added to the cells to kill any remaining extracellular bacteria and to prevent reinfection. At 24 h, the cells were washed with PBS and lysed in milliQ. Serial dilutions of the lysate were plated for determination of the number of intracellular CFU. Numbers of phagocytosed bacteria were determined by sampling at time zero and were found to be similar for all cells tested. In addition, binding and phagocytosis was evaluated by incubation of cells with FITC-labelled Salmonella bacteria, after which binding and uptake was determined by flow cytometry and confocal microscopy.
46 Acknowledgements We thank Andrea van Elsas, Pieter Leenen, William Nauseef, Michio Nakamura, Eric Brown and Mary Dinauer for providing antibodies, constructs and cells, Neil Barclay for sharing 2 unpublished data, Teunis Geijtenbeek for advice on the beads assay, and Elisabeth Eklund for her support and collaboration.
References 1. Roos,D., van Bruggen,R. and Meischl,C. (2003) 9. Jiang,P., Lagenaur,C.F. and Narayanan,V. (1999) Oxidative killing of microbes by neutrophils. Integrin-associated protein is a ligand for the P84 Microbes. Infect., 5, 1307-1315. neural adhesion molecule. J. Biol. Chem., 274, 559- 2. Bedard,K. and Krause,K.H. (2007) The NOX family 562. of ROS-generating NADPH oxidases: physiology 10. Seiffert,M., Cant,C., Chen,Z., Rappold,I., Brugger,W., and pathophysiology. Physiol Rev., 87, 245-313. Kanz,L., Brown,E.J., Ullrich,A. and Buhring,H.J. 3. Reeves,E.P., Lu,H., Jacobs,H.L., Messina,C.G., (1999) Human signal-regulatory protein is expressed Bolsover,S., Gabella,G., Potma,E.O., Warley,A., on normal, but not on subsets of leukemic myeloid Roes,J. and Segal,A.W. (2002) Killing activity of cells and mediates cellular adhesion involving its neutrophils is mediated through activation of counterreceptor CD47. Blood, 94, 3633-3643. proteases by K+ flux. Nature, 416, 291-297. 11. Vernon-Wilson,E.F., Kee,W.J., Willis,A.C., 4. Lindsey,S., Huang,W., Wang,H., Horvath,E., Zhu,C. Barclay,A.N., Simmons,D.L. and Brown,M.H. (2000) and Eklund,E.A. (2007) Activation of SHP2 protein- CD47 is a ligand for rat macrophage membrane tyrosine phosphatase increases HoxA10-induced signal regulatory protein SIRP (OX41) and human repression of the genes encoding gp91(PHOX) and SIRPalpha 1. Eur. J. Immunol., 30, 2130-2137. p67(PHOX). J. Biol. Chem., 282, 2237-2249. 12. Liu,Y., Tong,Q., Zhou,Y., Lee,H.W., Yang,J.J., 5. Zhu,C., Lindsey,S., Konieczna,I. and Eklund,E.A. Buhring,H.J., Chen,Y.T., Ha,B., Chen,C.X., Yang,Y. (2008) Constitutive activation of SHP2 protein et al. (2007) Functional elements on SIRPalpha IgV tyrosine phosphatase inhibits ICSBP-induced domain mediate cell surface binding to CD47. J. transcription of the gene encoding gp91PHOX during Mol. Biol., 365, 680-693. myeloid differentiation. J. Leukoc. Biol., 83, 680-691. 13. Lee,W.Y., Weber,D.A., Laur,O., Severson,E.A., McCall,I., Jen,R.P., Chin,A.C., Wu,T., Gernert,K.M. 6. Kautz,B., Kakar,R., David,E. and Eklund,E.A. and Parkos,C.A. (2007) Novel structural (2001) SHP1 protein-tyrosine phosphatase inhibits determinants on SIRP alpha that mediate binding gp91PHOX and p67PHOX expression by inhibiting to CD47. J. Immunol., 179, 7741-7750. interaction of PU.1, IRF1, interferon consensus sequence-binding protein, and CREB-binding 14. Hatherley,D., Graham,S.C., Turner,J., Harlos,K., protein with homologous Cis elements in the Stuart,D.I. and Barclay,A.N. (2008) Paired CYBB and NCF2 genes. J. Biol. Chem., 276, 37868- receptor specificity explained by structures of 37878. signal regulatory proteins alone and complexed with CD47. Mol. Cell, 31, 266-277. 7. Fujioka,Y., Matozaki,T., Noguchi,T., Iwamatsu,A., Yamao,T., Takahashi,N., Tsuda,M., Takada,T. 15. van Beek,E.M., Cochrane,F., Barclay,A.N. and van and Kasuga,M. (1996) A novel membrane den Berg,T.K. (2005) Signal regulatory proteins in glycoprotein, SHPS-1, that binds the SH2-domain- the immune system. J. Immunol., 175, 7781-7787. containing protein tyrosine phosphatase SHP-2 in 16. Barclay,A.N. and Brown,M.H. (2006) The SIRP response to mitogens and cell adhesion. Mol. Cell family of receptors and immune regulation. Nat. Biol., 16, 6887-6899. Rev. Immunol., 6, 457-464. 8. Adams,S., van der Laan,L.J., Vernon-Wilson,E., 17. Oldenborg,P.A., Zheleznyak,A., Fang,Y.F., Renardel,d.L., Dopp,E.A., Dijkstra,C.D., Lagenaur,C.F., Gresham,H.D. and Lindberg,F.P. Simmons,D.L. and van den Berg,T.K. (1998) Signal- (2000) Role of CD47 as a marker of self on red regulatory protein is selectively expressed by blood cells. Science, 288, 2051-2054. myeloid and neuronal cells. J. Immunol., 161, 1853- 18. Yamao,T., Noguchi,T., Takeuchi,O., Nishiyama,U., 1859. Morita,H., Hagiwara,T., Akahori,H., Kato,T.,
47 Inagaki,K., Okazawa,H. et al. (2002) Negative Siminovitch,K.A. and Downey,G.P. (2000) regulation of platelet clearance and of the Deficiency of Src homology 2-containing macrophage phagocytic response by the phosphatase 1 results in abnormalities in murine transmembrane glycoprotein SHPS-1. J. Biol. neutrophil function: studies in motheaten mice. J. Chem., 277, 39833-39839. Immunol., 165, 5847-5859. 19. Nikolic,T., de Bruijn,M.F., Lutz,M.B. and 28. Liu,Y., Buhring,H.J., Zen,K., Burst,S.L., Schnell,F.J., Leenen,P.J. (2003) Developmental stages of Williams,I.R. and Parkos,C.A. (2002) Signal myeloid dendritic cells in mouse bone marrow. regulatory protein (SIRPalpha), a cellular ligand Int. Immunol., 15, 515-524. for CD47, regulates neutrophil transmigration. J. 20. van Beek,E.M., de Vries,T.J., Mulder,L., Biol. Chem., 277, 10028-10036. Schoenmaker,T., Hoeben,K.A., Matozaki,T., 29. de Vries,H.E., Hendriks,J.J., Honing,H., De Langenbach,G.E., Kraal,G., Everts,V. and van Lavalette,C.R., van der Pol,S.M., Hooijberg,E., den Berg,T.K. (2009) Inhibitory regulation of Dijkstra,C.D. and van den Berg,T.K. (2002) Signal- osteoclast bone resorption by signal regulatory regulatory protein alpha-CD47 interactions are protein alpha. FASEB J., 23, 4081-4090. required for the transmigration of monocytes across 21. Kong,X.N., Yan,H.X., Chen,L., Dong,L.W., cerebral endothelium. J. Immunol., 168, 5832-5839. Yang,W., Liu,Q., Yu,L.X., Huang,D.D., Liu,S.Q., 30. Zhao,X.W., van Beek,E.M., Schornagel,K., Van der Liu,H. et al. (2007) LPS-induced down-regulation Maaden,H., van Houdt,M., Otten,M.A., Finetti,P., of signal regulatory protein {alpha} contributes to van Egmond,M., Matozaki,T., Kraal,G. et al. (2011) innate immune activation in macrophages. J. Exp. CD47-signal regulatory protein-alpha (SIRPalpha) Med., 204, 2719-2731. interactions form a barrier for antibody-mediated 22. Dong,L.W., Kong,X.N., Yan,H.X., Yu,L.X., Chen,L., tumor cell destruction. Proc. Natl. Acad. Sci. U. S. Yang,W., Liu,Q., Huang,D.D., Wu,M.C. and A, 108, 18342-18347. Wang,H.Y. (2008) Signal regulatory protein alpha 31. van der Loo,J.C., Slieker,W.A., Kieboom,D. negatively regulates both TLR3 and cytoplasmic and Ploemacher,R.E. (1995) Identification of pathways in type I interferon induction. Mol. hematopoietic stem cell subsets on the basis Immunol., 45, 3025-3035. of their primitiveness using antibody ER-MP12. 23. Stacey,K.J., Sester,D.P., Sweet,M.J. and Blood, 85, 952-962. Hume,D.A. (2000) Macrophage activation by 32. Kuijpers,T.W., vanBruggen,R., Kamerbeek,N., immunostimulatory DNA. Curr. Top. Microbiol. Tool,A.T., Hicsonmez,G., Gurgey,A., Karow,A., Immunol., 247, 41-58. Verhoeven,A.J., Seeger,K., Sanal,O. et al. (2007) 24. Zhen,L., King,A.A., Xiao,Y., Chanock,S.J., Orkin,S.H. Natural history and early diagnosis of LAD-1/ and Dinauer,M.C. (1993) Gene targeting of X variant syndrome. Blood, 109, 3529-3537. chromosome-linked chronic granulomatous 33. Heemskerk,M.H., Hooijberg,E., Ruizendaal,J.J., disease locus in a human myeloid leukemia cell line van der Weide,M.M., Kueter,E., Bakker,A.Q., and rescue by expression of recombinant gp91phox. Schumacher,T.N. and Spits,H. (1999) Enrichment Proc. Natl. Acad. Sci. U. S. A, 90, 9832-9836. of an antigen-specific T cell response by 25. Alblas,J., Honing,H., De Lavalette,C.R., retrovirally transduced human dendritic cells. Cell Brown,M.H., Dijkstra,C.D. and van den Berg,T.K. Immunol., 195, 10-17. (2005) Signal regulatory protein alpha ligation 34. Kinsella,T.M. and Nolan,G.P. (1996) Episomal induces macrophage nitric oxide production vectors rapidly and stably produce high-titer through JAK/. Mol. Cell Biol., 25, 7181-7192. recombinant retrovirus. Hum. Gene Ther., 7, 1405- 26. Dong,Q., Siminovitch,K.A., Fialkow,L., 1413. Fukushima,T. and Downey,G.P. (1999) Negative 35. Lindberg,F.P., Gresham,H.D., Schwarz,E. and regulation of myeloid cell proliferation and Brown,E.J. (1993) Molecular cloning of integrin- function by the SH2 domain-containing tyrosine associated protein: an immunoglobulin family phosphatase-1. J. Immunol., 162, 3220-3230. member with multiple membrane-spanning 27. Kruger,J., Butler,J.R., Cherapanov,V., Dong,Q., domains implicated in alpha v beta 3-dependent Ginzberg,H., Govindarajan,A., Grinstein,S., ligand binding. J. Cell Biol., 123, 485-496.
48
Engagement of SIRPα inhibits growth and induces programmed cell death in 3 acute myeloid leukemia cells Mahban Irandoust1,2*, Julian Alvarez Zarate3*, Isabelle Hubeek1,2, Ellen M. van Beek3, Karin Schornagel3, Aart J.F. Broekhuizen1 , Mercan Akyuz1, Arjan A. van de Loosdrecht2, Ruud Delwel4, Peter J. Valk4, Edwin Sonneveld5, Pamela Kearns6, Ursula Creutzig7, Dirk Reinhardt7 , Eveline S.J.M. de Bont 8, Eva A. Coenen 9, Marry M. van den Heuvel-Eibrink9, C. Michel Zwaan9, Gertjan J.L. Kaspers1, Jacqueline Cloos1,2§, Timo K. van den Berg3§
Departments of 1Pediatric Hematology/Oncology and 2Hematology, VU University Medical Centre, Amsterdam, The Netherlands 3Sanquin Research & Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. 4Dept of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands. 5Dutch Childhood Oncology Group (DCOG), The Hague, The Netherlands. 6School of Cancer Sciences, University of Birmingham, Birmingham, UK. 7Department of Pediatric Hematology/Oncology, Medical School Hannover, Hannover, Germany. 8University Medical Center Groningen, Groningen, The Netherlands. 9Dept of Pediatric Hematology/Oncology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands.
* and §: These authors contribute equally to this manuscript.
PLoS One. 2013;8(1):e52143
52 Abstract Background: Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia. 3 Design and Methods: We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.
Results: By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0-M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs. Conclusions: Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.
53 Introduction Currently only one third of adult patients diagnosed with acute myeloid leukemia (AML) can be cured despite aggressive chemotherapy, and relapse rate is still high in these patients [1,2,3]. Although the prognosis of pediatric AML patients is better, the outcome remains relatively poor. With standard induction chemotherapy, complete remission (CR) for newly diagnosed pediatric AML is achieved on more than 80% of patients, however, about 30-50% of these children relapse from minimal residual disease (MRD) cells that apparently survived chemotherapy [4,5,6]. Therefore, new treatment modalities for AML are warranted. Distinct morphological subgroups in French-American-British (FAB) classification associate with different chromosomal rearrangements and acquisition of recurring genetic abnormalities; for example t(8;21) )(q22;q22) and t(15;17) )(q22;q21) create fusion genes, AML/ETO and PML/ RARα, which predominate in FAB M2 and M3 AML subtypes respectively. These proteins are two of the most common AML-associated oncofusion proteins, which in total represent 20% of the AML occurrence [7]. The cytogenetic rearrangement involved with t(8;21) disrupt genes that are required for normal hematopoietic development, such as subunits of core-binding factor [8]. Expression of the PML/RARα fusion protein leads to a differentiation block at the promyelocytic stage that can be relieved by all-trans-retinoic acid (ATRA). Studies on AML/ETO and PML/RARα expressing cells have revealed that aberrant signaling pathways are involved [9]. Insights into these signaling pathways in AML at molecular level will pave ways for new treatment modalities. Signal regulatory protein alpha (SIRPα) is a transmembrane receptor composed of 3 immunoglobulin-like domains in its extracellular region and an intracellular domain containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) which recruit and activate SHP-1 and SHP-2 [10,11]. SIRPα is predominantly expressed on myeloid and neuronal cells [10] and its activation has been implicated in regulation of different cellular functions such as adhesion, migration, growth and differentiation [12,13,14]. Despite restricted expression of SIRPα, CD47, the natural ligand for SIRPα [15], is ubiquitously expressed and interacts with the SIRPα extracellular region. This interaction results in inhibition of phagocytosis by macrophages through tyrosine phosphatase activation and inhibition of myosin accumulation [16,17,18]. CD47 functions as a ‘‘don’t eat me’’ signal and plays a key role in the programmed cell removal of abberant versus normal cells [19]. Indeed, it was recently shown that CD47 is overexpressed on AML leukemic stem cells as compared to their normal counterparts (hematopoietic stem cells) and this contributes to inhibition of phagocytosis and clearance of LSCs [20]. In addition, blocking antibodies directed against CD47 promoted phagocytosis as suggested through disruption of CD47-SIRPα interaction and this enhanced tumor clearance in vivo [20,21,22]. These findings are in line with several studies, which reported elimination of tumor cells by employment of CD47 blocking antibodies [20,22,23,24,25,26,27]. Although SIRPα is known to be expressed by AML cells as well [11,28], its function on these cells has not been identified. Furthermore, previous studies were performed with antibodies that not
only recognized SIRPα, but also several of the related molecules such as SIRPβ1 and SIRPγ. In the present study we have examined the mRNA and protein expression of SIRPα in a large cohort of
54 AML patients and determined its relevance for AML cell survival. We show for the first time that SIRPα ligation triggers programmed cell death in AML cells and synergizes with antileukemic agents.
Design and Methods Antibodies and drugs 3 At this moment no human agonistic SIRPα antibody is available. However, a rat agonistic SIRPα antibody (ED9) was generated in our laboratory [10,29], that is also commercially available at Serotec (Oxford, UK). Such an agonistic antibody has much higher affinity to rat SIRPα as compared to CD47-Fc fragments [30,31], so the use of this agonistic antibody was preferred for mechanistic studies and optimal SIRPα triggering. To be able to exert an agonistic signal using the available rat antibody, a chimeric construct of SIRPα was generated carrying rat SIRPα extracellular domain and human transmembrane and cytoplasmic region. The following monoclonal antibodies (mAb) were used in this study: ED9 (anti-rat SIRPα; mouse IgG1 isotype) was labeled with Alexa-633, which was obtained from Invitrogen (Breda, The Netherlands). Considering the differences between rat and human SIRPα, it is not likely that the ED9 agonistic antibody cross-reacts with the human SIRPα [30,31]. Rabbit polyclonal Ab8120 (Abcam, Cambridge, United Kingdom) is directed to the cytoplasmic tail of human SIRPα. Mouse anti- actin monoclonal antibody, mAb1501R, was purchased from Chemicon International (Temecula, CA, USA). mAb against caspase-3 was obtained from Cell Signaling Technology (Boston , MA, USA). APC-labeled anti-human CD11b was acquired from BD pharmingen (San Jose, CA, USA). PE- labeled B6H12 was purchased from Santa Cruz Biotechnology and B6H12 F(ab’)2-fragments were generated in our laboratory by pepsin digestion as previously described [32]. The histone deacetylase (HDAC) inhibitors (Trichostatin A (TSA), valproic acid (VPA) and sodium butyrate) and ATRA were purchased from Sigma Aldrich (St Louis, MO, USA). 5-aza- 2-deoxycytidine (DAC, decitabine) was kindly provided by Pharmachemie BV (Haarlem, The Netherlands). Cytarabine (Cytosar®) was obtained from Pharmacia & Upjohn (Woerden, The Netherlands). Daunorubicin (Cerubidine®) was purchased from Rhone Poulenc Rorer (Amstelveen, The Netherlands). Etoposide (PV16, Vepesid®) was obtained from Bristol-Myers Squib (Woerden, The Netherlands). Imatinib was provided by Novartis (The Netherlands). zVAD was obtained from Merk Biosciences (Darmstadt, Germany).
Patient samples For the expression array experiments bone marrow and/or peripheral blood samples were collected from adult AML patients at diagnosis, as described by Valk PJ et al. [33]. Bone marrow and/or peripheral blood samples from children diagnosed with de novo AML were collected from the following study centers: VU University Medical Center, Amsterdam, The Netherlands; The Dutch Childhood Oncology Group (DCOG), The Hague, The Netherlands and the AML BFM-study Group, Hannover, Germany. AML subtypes were classified according to the criteria by Bennett et al., including the modifications to diagnose FAB subtypes [34]. Mononuclear cells were isolated by density gradient centrifugation as described previously [35]. All samples
55 contained at least 80% leukemic cells, as determined morphologically by analyzing May- Grünwald-Giemsa (Merck, Darmstadt, Germany) stained cytospins.
Cell Lines and culture conditions The human leukemic cell lines KG1a (primitive human hematopoietic myeloid progenitor), Kasumi-1 (human acute myeloid leukemia, FAB M2 t(8;21)), HL-60 (human promyelocytic leukemia), NB4 (human acute promyeloctic leukemia, FAB M3 t(15;17)),U937 (human acute monocytic leukemia), THP-1 (human acute monocytic leukemia), CEM (human acute lymphoblastic leukemia), Jurkat (human T-cell acute lymphoblastic leukemia) were routinely cultured in RPMI 1640 medium (Gibco Laboratories, Irvine, UK) supplemented with 10% fetal calf serum (Integro BV, Dieren, the Netherlands). Kasumi-1 cells (0,5x106) were incubated for 0, 3, 24, 48, 72 and 96 hours with 1 µM
DAC, 0,3 µM TSA, 0,5 mM VPA and 1 mM sodium butyrate in 5% CO2 humidified air at 37ºC. Cells were subsequently used for Western Blot analysis, as described below. Human neutrophils were isolated from heparinized blood of healthy individuals by centrifugation over isotonic Percoll (Pharmacia, Uppsala, Sweden) and subsequent lysis of erythrocytes as described [36]. Neutrophils were cultured in Hepes-buffered saline solution supplemented with 1% human serum albumin (Cealb: Sanquin, Amsterdam, the Netherlands) and 5 mM glucose.
DNA isolation Cell lines: 1-5x106 of KG1a, Kasumi-1 and HL-60 cells were resuspended in HIRT buffer (0,6% SDS, 10 mM Tris, 10 mM EDTA pH 8). Proteinase K was added and samples were incubated at 50°C for 2 hours and subsequently at 37°C overnight. Phenol: chloroform (1:1) was added and the solution was mixed vigorously and centrifuged. The aqueous layer was transferred into a tube and the phenol/chloroform extraction was repeated. Following centrifugation, the aqueous layer was removed and the DNA was precipitated by NaOAC/EtOH (1:24), washed with 70% EtOH and resuspended in TE buffer. DNA concentrations were measured with spectrophotometer (Nanodrop, Isogen, The Netherlands). Patient samples: DNA was isolated from cryopreserved cytospins. A sterile swab and a drop of sterile water were used to wipe the cells from the slides. The swab was transferred into buffer (100 mM Tris, 10 mM NaCl, 5mM EDTA, 1% SDS pH 9) containing proteinase K and incubated overnight at 52°C. Tubes were centrifuged and DNA was isolated as described above.
Western blot analysis Cells were washed in PBS, centrifuged and the cell pellet was lysed with Igepal lysis buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche). Whole cell lysates were clarified by centrifugation and denatured in Laemmli’s sample buffer (Bio-rad Laboratories, Hercules, CA, USA). After that cell lysates were subjected to Western blot and membranes stained with primary and secondary antibodies.
56 Construction of retroviral vectors and transduction of Kasumi-1 cells The rat-human SIRPα fusion construct (chSIRPα) was generated from cDNA and PCR fragments as follows: nucleotide 1-1236 of the rat SIRPα cDNA [10] was fused to nucleotide 1230-1509 of the human cDNA (prot. accession No: NM_080792). The chSIRPα protein contains amino acids 1-412 (rat extracellular domain) and amino acids 411-503 (human transmembrane and cytoplasmic region) resulting in a total length of 505 amino acids, including the signal sequence. 3 The sequence of the construct was confirmed by automated sequencing. For retroviral transduction the chSIRPα construct was cloned into the retroviral expression vector pLZRSpMBN-linker-IRES-eGFP(NotI) [37]. The Phoenix-A packaging cell line [38] was transfected with the retroviral construct containing chSIRPα or empty vector (EV) containing eGFP by calcium-phosphate transfection. After puromycin selection (Sigma-Aldrich, St Louis, MO, USA), harvested virus supernatant [37] was used for transduction of Kasumi-1 or NB4 cells. chSIRPα-expressing cells were subsequently selected in several rounds by FACS sorting (MoFlo,Dako Cytomation) on the basis of eGFP expression to reach to >98% positive cells. Cell surface expression of chSIRPα was determined by FACS analysis using the ED9 mAb, as described below. FACS-sorted, mock-transduced cells containing EV were used as controls in all experiments. Ectopic expression of SIRPα and/or eGFP were regularly monitored and were found to be stable for several months, with >90% positive cells.
Cell proliferation and programmed cell death For cell proliferation assays cells were seeded in triplicate at 1x105/ml concentration in 96-well plates and treated with 10 µg/ml of the ED9 mAb where indicated and incubated for the mentioned time points up to 7 days. For programmed cell death (PCD) experiments, cells were seeded at 0,5x106 cells/ml and on days 1 and 3 PCD was measured by Annexin V-phycoerythrin and 7-amino-actinomycin D (7-AAD) or DAPI double staining according to the manufacturer’s protocol (BD Pharmingen, San Jose, CA, USA). All analyses were performed on a FACS calibur (BD Biosciences, San Jose, CA, USA). Cells positively stained with Annexin-V and 7-AAD negative were considered to be early apoptotic.
Growth inhibition studies Growth inhibitory effects of chemotherapeutics in combination with ED9 mAb were evaluated with the MTT-assay, as described previously [39]. SIRPα and EV cells were incubated with 4 concentrations of cytarabine (ara-C) 2,5 – 0,002 µM, daunorubicin (DNR) 18,0 – 0,005 µM, etoposide (VP16) 4,4 – 0,01 µM and imatinib 5 - 0,005 µM in combination with one fixed concentration of the ED9 mAb (10 µg/ml); these experiments were done in triplicate. Within each experiment all drugs were tested alone, as well as in combination. Drug interactions between chemotherapeutic drugs and ED9 mAb were studied by using the multiple drug effect analysis of Chou and Talalay [40] (Calcusyn software, Biosoft, Cambridge, UK) and antagonistic, additive or synergistic interactions were determined. This method is commonly used in many drug interaction studies [41]. The interactions are determined by Combination Index (CI) which
57 indicates synergism (CI<0,9), additivity (CI=0,9-1,1) or antagonism (CI>1,1). In the CI-FA plot the CI values > 0,5 are evaluated and per experiment a mean CI was calculated from FA values 0,5, 0,75 and 0,9. The average CI (± SD) of three experiments is given for each of the combinations.
Bisulphite sequencing Kasumi-1 cells and the 4 t(8;21) AML patient samples were bisulphate treated.[63] PCR amplification was performed using the following primer sets: PTPNS1-F 5’–GTA TTT ATT TTT AAG AGG GGT TTT TA-3’and PTPNS1-R 5’ CAAACTTATTTTTCTAAAATCAA-3’ in a two round PCR approach. PCR products were extracted from agarose gel by Qiagen gel-extraction kit (Qiagen Benelux BV) and cloned into the pGEM®-T Easy Vector Systems according to manufacturer’s protocol (Promega, Madison, WI, USA). After overnight incubation at 37°C clones were picked and cultured in liquid cultures at 37°C. The Qiagen DNA mini-prep kit (Qiagen Benelux BV) was used to extract DNA and the desired inserts were enzymatically cleaved from the pGEM®-T Easy Vector to check if the right insert was cloned. BaseClear BV performed automated DNA sequencing
Statistical analysis The statistical significance of measured differences in proliferation and PCD between the various conditions and cell populations was determined using the paired Student’s t-test. Calculations were performed using Graph-pad Prism and SPSS software. Statistical analysis (Cox proportional hazards model; reported p values corresponded to the Wald test) on overall survival and event free survival was performed in SPSS software. Survival distribution was compared with median SIRPα expression of the whole group.
Results SIRPα mRNA expression in AML The mRNA level in pediatric AML patients was analyzed in a micro-array dataset containing 226 samples [42]. SIRPα mRNA expression varied considerably among different AML patients (Figure 1A). A clear association was observed between SIRPα expression and AML FAB subtypes with the highest levels found in the myelo-monocytic FAB M4/M5 subsets. The SIRPα expression levels in myeloblastic leukemic blasts were relatively low in the FAB M0-M3 subtypes. The M6 erythroid type of AML also showed a low SIRPα expression. Comparing acute myeloid leukemia cases with normal bone marrow, lower expression of SIRPα was observed in immature AML subtypes (Figure 1A), suggesting a myeloid differentiation stage-dependent expression, which is in line with the high expression of SIRPα found on normal monocytes and macrophages [10]. In particular, the significant difference between M0-M3 and M4/M5 (p <0,001), and the different expression distribution are depicted in Figure 1B. Classifying pediatric samples based on karyotypes showed the highest (p=1,63x10-10) and lowest (p=2,05x10-6) expression of SIRPα in MLL-rearranged and t(8;21) subgroups, respectively (Figure 1C). Since we only accessed one pediatric dataset, we validated these findings in adult AML datasets [33,43,44] and consistent
58 with the pediatric results the highest level of SIRPα expression was found in M4 and M5 subtypes as compared to the immature groups such as FAB M0, M1, M2 and M3 (Figure 2). In addition, karyotype classification of 285 AML patients in an adult dataset [33], showed increased SIRPα expression in inv(16), and MLL-rearrangement groups (depicted as clusters 5, 9 and 16) in comparison to t(8;21) and t(15;17) AML (Figure 2B). To examine whether SIRPα expression is correlated with patient survival, we performed an 3 analysis on overall survival (OS) and event free survival (EFS) in the pediatric cohort (n=175), for which follow-up data are available. We observed that higher SIRPα expression compared to the median of the 175 patients (8,1 arbitrary units), significantly correlated with unfavorable outcome. Figure 1D shows the Kaplan Meier analysis based on OS (log-rank p=0,024, hazard ratio (HR) 1,7, p=0,026). For EFS the data were similar: log-rank p=0,029, and HR: 1,5, p=0,031. In addition, following stratification on karyotype, we could not generally find any significant relation between SIRPα and outcome (not shown). Only within the MLL rearranged samples a high SIRPα expression (above the median SIRPα expression of the MLL rearranged group) exhibited a trend towards unfavorable outcome (HR: 2,28, p=0,062). It is difficult to compare the outcome between children and adults since the adults have, in general, a very dismal prognosis. In the data set of Valk et al. [30] no association was found between SIRPα expression and outcome on the whole cohort. For the adult MLL rearranged samples, high SIRPα levels associated with a slightly favorable outcome (HR: 0,84, p=0,027). In both children and adults, multivariate analysis reveals that SIRPα is not an independent risk factor. Recent studies have shown that CD47, the ligand for SIRPα, is a prognostic factor in breast cancer and its expression correlates with SIRPα expression in bone marrow and peripheral blood of breast cancer patients [32,45]. We therefore evaluated a possible association between SIRPα and CD47 expression in different datasets [33,42,46], however we did not find any evidence for such an association in AML. Figure 1E shows that CD47 is equally distributed among the pediatric AML subtypes while there is a clear difference in SIRPα expression with the MLL- rearranged clustering in the high SIRPα range.
SIRPα protein expression in AML We determined SIRPα protein expression, by Western blotting using an antibody directed to the cytoplasmic tail of the human SIRPα on various leukemic cell lines and patient samples. While no expression was observed among acute lymphoblastic leukemia (ALL) cell lines, AML cell lines differentially expressed the SIRPα protein (Figure 3A). In particular, immature myeloblasts such as t(8;21) Kasumi-1, KG-1, HL60 cells or promyelocytes like t(15;17) NB4 cells expressed low or undetectable levels of SIRPα protein compared to more differentiated monocytic cells such as THP-1 and U937 (Figure 3A). We also analyzed 20 primary pediatric AML patient samples and consistent with the mRNA data, SIRPα protein expression was low/undetectable in immature subgroups compared to the more mature groups such as M4 and M5 (Figure 3B, C). As expected we did not observe SIRPα expression in ALL patient samples (n=10) (Figure 4). Collectively, these findings suggest a selective myeloid and differentiation stage-dependent expression of SIRPα mRNA and protein expression in AML.
59 A PediatricPediatric AML AML samplessamples (n=226)(n=226) Expression Expression α α (arbitrary units) (arbitrary units) SIRP SIRP SIRP
M0 M1 M2M2 M3M3 M4M4 M5M5 M6 M6 Other Other ND ND
AMLAML FABFAB subtypessubtypes
B ▶
Figure 1. SIRPα mRNA expression and its prognostic effect in pediatric AML cohort. (A) SIRPα mRNA expression was determined in different FAB subtypes. The dots represent individual patients and the horizontal bar is the group mean. The horizontal dotted line represents the mean levels (139,6; n=5) of SIRPα expression in normal CD34+ HSC. (ND: not determined). (B) Frequency of SIRPα expression among different FAB subtypes of AML patients is shown as stacked histograms. (C) SIRPα mRNA expression as stratified after karyotype (NK: normal karyotype). (D) Overall survival of pediatric AML patients (n=175) stratified according to either low (< median of 8,1) or high (≥ median 8,1) SIRPα mRNA expression. (E) Correlation between CD47 and SIRPα mRNA expression is shown by the blue and red dots, respectively. The lower bar demonstrates the cluster of patients in karyotypes, in which higher SIRPα expression is clustered in blue, representing the MLL rearrangement group on the right side. ▶
60 C Pediatric AML samples (n=226)
3 Expression α Expression (arbitrary units) SIRP
11q23 t(8;21) inv(16) t(15;17) t(7;12) NK Other ND
D
p log rank = 0.024
Low SIRPα expression
High SIRPα expression
Overall survival (months)
E 2 log of CD47 log log of SIRPA 2
Samples ordered by SIRPα
Karyotype
61 A Adult AML samples (n=285) Expression α (arbitrary units) SIRP
M0 M1 M2 M3 M4 M5 M6 ND AML FAB subtypes
(%) pe ity (%) al oty rm ary k B bno al ormality a n the norm th on ab i m n w p en with me m rou ci g eci pe p Sub Most com S S 1 t(11q23) 43 43 2 FLT3 ITD 82 76 3 FLT3 ITD 53 68 4 CEBPA 53 67
5 61
6 FLT3 ITD 100 88 7 67 8
9 inv(16) 83
10 EVI1 45 11 78 12 t(15;17) 95
13 t(8;21) 100 14 15 CEBPA 63 63 16 t(11q23) 45 Patients with AML (n=285)
-100% +100% Pearson’s Correlation Coefficient
Figure 2. SIRPα mRNA expression in adult AML cohort. (A) SIRPα mRNA expression was determined in different FAB subtypes of 285 adult patients. The dots represent individual patients and the horizontal bar is the mean of the group (ND: not determined). (B) Adapted correlation view of the 16 unsupervised clusters (indicated on the left) of 285 adult AML specimens identified by mRNA profiling, [33] including the expression levels of SIRPα using 3 independent probes on the right diagonal axes. SIRPα expression is high in clusters 5, 9 and 16, but low in most other clusters, including clusters 12 and 13, which contain almost exclusively t(15;17) and t(8;21) AML, respectively.
Upregulation of SIRPα upon differentiation of t(15;17) AML cells To address whether SIRPα expression is upregulated upon differentiation of AML cells, we selected the NB4 cell line, a t(15;17) M3 FAB subtype, which only express low levels of SIRPα (Figure 3A). Since ATRA treatment of t(15;17) APL patients is known to result in granulocytic differentiation [47,48], we examined if SIRPα expression increased after exposure to ATRA. To address this, the
62 A
CEM Jurkat Kasumi-1 HL-60 NB4 U937 THP-1 KG-1 SIRP α
β -Actin 3
AML cell lines ALL cell lines
B AML patient samples
SIRPα
β-Actin M1 M3 M4 M5 M0 M2 M5 NBM CD34+
AML patient samples SIRPα
β-Actin
M2 M5 M4 M4 M4 M4 M1 M5 M1 M2 M4
M4 inv16 M4 inv16
C 800 700 600 500 400 300 expression (Arbitrary units) (Arbitrary expression
200 100 RelativeSIRP 0 M0 M1 M1 M2 M2 M2 M3 M4 M4 M4 M4 M4 M4 M5 M5 M5 M5 M1 NBM M4INV M4INV CD34+ M0-M3 M4-M5
AML FAB subtypes
Figure 3. SIRPα protein expression in AML cell lines and patients. Western blot analysis was performed in (A) cell lines and (B) 20 pediatric AML patient samples. β-actin staining was used as loading control. (C) SIRPα expression is quantified relative toβ -actin expression.
NB4 cells were incubated with 1µM ATRA for 7 consecutive days and granulocytic differentiation of the NB4 cells was confirmed by upregulation of the common myeloid marker CD11b (Figure 5A). In concert with the increased differentiation of NB4 cells, SIRPα protein expression was markedly upregulated following ATRA exposure (Figure 5B). This upregulation was already detectable after 24 hrs and it was further increased during the following days of treatment.
63 64 cell surface expressioncell surface of the common myeloid marker, CD11b. (B) SIRP triggering. (A) NB4 cells were to exposed 1 µM ATRA and granulocytic differentiation of the cells was by examined containing the extracellular region of CD47, the natural ligand of SIRP analysis ofanalysis chSIRP chSIRP SIRP patient samples by western blotting showed that SIRP by westernpatient blotting samples showed proliferation (Figure 6). In order to investigate of SIRP the effect used as a loading control. Figure 5. Upregulation of SIRP [49]antibodies against rat SIRP Our initial experiments with the rat that myeloid several cell line NR8383 showed agonistic monoclonal SIRP Induction ofprogrammedcelldeathint(15;17)AMLcellsfollowing Figure 4. SIRP cells showed that the vast majoritycells showed (>90%) transfected of cells had been by chSIRP by retroviral transduction. cytrometric Flow of analysis the retrovirally transduced and FACS-sorted [28,32]. In addition CD47-Fc[28,32]. did a sufficiently not show high affinity for binding to SIRP specificity, cross-reactivity showing with other or SIRPlacked family members, the agonistic activity mAb, on rat macrophages or granulocytes ([10] and data not shown). cell death (PCD) as quantified by flow cytometry using annexin-V/ (PCD)cell death cytometry as quantified by flow staining5C). (Figure 7-AAD of chSIRPlevels of SIRP vitro after exposure to 1 µM ATRA inis combinationshown with 10µg/ml of ED9 (*: significant difference EV transduced NB4 cells was quantified by APC-Annexin V and PE-7AAD staining.FACS (E) Percentage of apoptosis a variety of previously against reported SIRP antibodies the extracellular region of rat SIRP enabled theenabled use of the rat SIRP specific blotting, is upregulated in ATRA-incubated NB4 cells. Ligation of SIRP α experiments to used as an be agonistic (data not Hence, shown). to able to be study the effect α [10]. Stable t(15;17) NB4 expressing cell lines chSIRP α α expressing cells. (D) 24 ED9 following (10µg/ml) hrs incubation, the percentage of in cell chSIRP death triggering in human myeloid generated cells, we a chimeric SIRP ligation α is not expressed of protein in Analysis ALL patient samples. expression of SIRP α α expression (i.e. fluorescence) mean were comparable to those same with theseen, surface expression surface is determined by using in transduced ED9 mAb NB4 empty vector and α in NB4 cells by agonistic resulted ED9 mAb in of induction programmed α upon differentiation upon of t(15;17) NB4 cells and of itsinduction following cell death SIRP -actin α (for example ED9, ED17 or OX41) or recombinant Fc-fusion proteins α α and the transmembrane and the cytoplasmic domains of human THP-1
α CCRF-CEM agonistic ED9 mAb. This chSIRP 1 2 3 4 5 6 7 8 9 10 9 8 7 6 5 4 3 2 1 L ain samples ALL patient α β is not expressed in these samples. -actin is used as a loading control. cytometric (C) Flow α , but these either lacked the appropriate α or empty vector (EV) were generated α protein expression, determined by western α ligation in NB4 tested cells we α α , are able to suppress cell α (chSIRP construct consisted construct of α β α -actin staining was (Figure 5C). The (Figure The 5C). α ) construct that ) that construct n pedi in p <0,05). α in in our in ti ALL atric α and ▶ A +ATRA - ATRA
3
B 1μM ATRA Days: 012357
SIRPα
-actin
EV chSIRPα C
NB4
SIRP α
D EV chSIRPα
control 7-AAD ED9
Annexin-V
E * * % apoptotic cells apoptotic %
EV chSIRPα
65 control
OX41
ED17
ED9
CD47-Fc
0 20 40 60 80 100 120 Proliferation (relative 3H-thymidine incorporation)
Figure 6. Triggering SIRPα in the rat NR8383 macrophage cell line inhibits proliferation. NR8383 cells were incubated for 18 hours with CD47-Fc protein or indicated anti-rat SIRPα monoclonal antibodies (ED9, ED17 or OX41). 3H-thymidine was added for 4 hours and proliferation was determined by incorporated radioactivity.
After 24h of exposure to ED9 mAb, the percentage of annexin-V positive cells was significantly higher in the NB4 chSIRPα cells (47,3 ± 8,6%), as compared to NB4 EV cells (15,1 ± 5,0%; p=0,009) (Figure 5C). These data support the requirement for ED9 binding to SIRPα, since no induction of cell death was observed in NB4 EV cells. These findings provide evidence for induction of cell death capacity by SIRPα triggering in APL cells. From a therapeutic perspective, it would appear beneficial to relieve differentiation block in M3 AML cells by using ATRA and as a result to upregulate SIRPα expression, which can subsequently be targeted by an agonistic antibody to induce PCD. Clearly prerequisite for such a strategy to be successful would be the efficiency of SIRPα triggering following differentiation. Therefore we examined whether apoptosis induction via SIRPα persisted after differentiation with ATRA. Stably transduced NB4 (chSIRPα and EV) cells were exposed to 1µM ATRA in combination with the fixed concentration of 10µg/ml ED9 mAb, which was shown to trigger PCD in the NB4 chSIRPα cells. As expected, ATRA treatment alone did not have any effect on PCD, whereas ED9 resulted in PCD in the chSIRPα transduced NB4 cells and this was not significantly altered after differentiation with ATRA. Furthermore while SIRPα triggering by ED9 mAb does induce programmed cell death, we found that it did not affect differentiation (Figure 7).
EV chSIRPα
CD11b
Figure 7. NB4 cells differentiate by ATRA exposure. Differentiation of NB4 cells stably expressing chSIRPα and EV was examined by flow cytometry after treatment with ATRA or ED9. increased expression of CD11b was observed only after ATRA but not by ED9 treatment.
66 Taken together, these data indicate that ATRA provides a stimulus for differentiation of t(15;17) APL cells and this results in upregulation of SIRPα expression to a level that makes the cells prone to cell death induction via SIRPα triggering even in differentiated M3 cells.
Upregulation of SIRPα following differentiation of t(8;21) AML cells To examine whether the low endogenous expression of SIRPα is also upregulated following 3 differentiation of other low-SIRPα-expressing myeloid leukemic cells, we selected t(8;21) Kasumi-1 cells. These cells belong to AML M2 FAB subtype and express low endogenous levels of SIRPα (Figure 3A). It has been shown that histone deacetylase (H-DAC)-inhibitors such as butyrate, valproic acid (VPA) and trichostatin (TSA) and DNA methyltransferase (DNMT)- inhibitors such as decitabine, induce granulocytic maturation of t(8;21) acute myeloid leukemia cells [50,51,52,53] (and data not shown). Kasumi-1 cells were exposed to TSA, VPA,butyrate or decitabine for indicated time points, which resulted in markedly increased SIRPα levels (Figure 8). With all drugs, an increase in SIRPα protein expression was detected as early as 3 hrs after exposure and this expression reached maximal levels after approximately 24 hrs. These data show that SIRPα is upregulated following differentiation of Kasumi-1 cells. An alternative explanation for the upregulation of SIRPα in t(8;21) AML was that these inhibitors of epigenetic silencing had acted directly on the SIRPα gene (accession number:
Decitabine (1 µM) Valproic acid (0.5 mM)
SIRPα SIRPα
-actin -actin
0 3 24 48 72 96 (hours) 0 3 24 48 72 96 (hours)
Butyrate (1 mM) Trichostatin A (300 nM)
SIRPα SIRPα
-actin -actin
0 3 24 48 72 96 (hours) 0 3 24 48 72 96 (hours)
Figure 8. SIRPα upregulation in t(8;21) Kasumi-1 cells following treatment with inhibitors of epigenetic gene silencing. Kasumi-1 cells were incubated with 1 µM Decitabine, 0,5 mM valproic acid, 1 mM Butyrate and 300 nM Trichostatin. Endogenous SIRPα protein level, determined by Western blotting was upregulated at indicated time points. β-actin staining was used as a loading control.
67 NP-542970.1), and in fact this seemed possible since a prominent CpG island is present in the PTPNS1 promoter region. DNA methylation in this region was explored in Kasumi-1 cells and four t(8;21) AML patients by bisulphite DNA sequencing. Results revealed actually very low levels of DNA methylation in the promoter region (Figure 9). We also analyzed the SIRPA2p pseudogene, in which abundant methylation was detected in the corresponding region [49]. Taken together these data strongly suggest that the increased level of SIRPα in t(8;21) Kasumi-1 cells following demethylating agents is the result of differentiation.
Inhibition of proliferation and induction of PCD in t(8;21) AML cells by SIRPa triggering Since triggering SIRPα by agonistic ED9 mAb induced cell death in t(15;17) NB4 cells, we extended our findings in t(8;21) Kasumi-1 cells, stably transduced with chSIRPα or EV (Figure 10A). The overexpression of chSIRPα in Kasumi-1 cells itself did not affect the growth, however culturing
Kasumi-1 SIRPα #B SIRPα
Kasumi-1 SIRPα2p #B SIRPα2p
#C SIRPA
SIRP?
CpG island MSP2 #A SIRPMSPα Primers MSP1 F1/R1 F3/R4 Sequence primers #D SIRPα
#A SIRPα2p
Figure 9. PTPNS1 promoter region is not methylated. Each circle indicates a CpG dinucleotide (open circles: unmethylated, filled circles: methylated) and each line represents analyses of a single amplified clone. SIRPα2p pseudogene, which is highly highly homologous to PTPNS1 was used as a positive control with high degree of methylation.[49] Methylation specific PCR and bisulphate sequencing [63] of the Kasumi-1 cell line and four t(8;21) AML patients did not reveal methylation of the PTPNS1 promoter region.
68 Kasumi-1 chSIRPα cells in the presence of the agonistic rat ED9 antibody caused a significant inhibition of proliferation (Figure 11). In order to investigate whether growth suppression triggered by chSIRPα ligation coincided with an enhanced level of cell death induction, the percentage of cell death was quantified by flow cytometry, using Annexin V and 7-AAD staining (Figure 10B). Already 24 hours after adding ED9 mAb the percentage of early dying cells, defined as Annexin V positive, was significantly higher in the Kasumi-1 chSIRPα cells, as 3 compared to Kasumi-1 EV cells, while addition of an irrelevant antibody had no effect (Figure 10B, lower panel). Similar results were observed on day 3 of treatment, at which ligation with ED9 mAb had caused significant cell death in Kasumi-1 chSIRPα cells (35,2 ± 15,3% versus 10,4 ± 2,8% in untreated control cells; p=0,02). All effects required ED9 binding to chSIRPα, since no induction of cell death was observed in Kasumi-1 EV cells. We next investigated whether cell death induction in Kasumi-1 cells was involved caspase activity, which is often required for PCD. First, we investigated activation of the effector caspase- 3, which can be measured by evaluating the appearance of the p17 caspase-3 cleavage product. As shown in Figure 10C triggering of SIRPα in Kasumi-1 chSIRPα cells did not result in any detectable caspase-3 cleavage, whereas this was detected upon culture of freshly isolated neutrophils. Furthermore, incubation with the universal inhibitor of caspases, zVAD (10μM), did not affect programmed cell death induction via SIRPα in Kasumi-1 chSIRPα cells whereas neutrophil apoptosis was zVAD sensitive (results not shown). Collectively, these findings indicate a growth- suppressive and caspase-independent mode of PCD induction via SIRPα in t(8;21) AML. To examine whether the observed effects of ED9 mAb in Kasumi-1 cells, occur through blocking of CD47-SIRPα interactions, we used the blocking anti-CD47 antibody B6H12. As shown in Figure 12, induction of cell death by ED9 cannot be mimicked by B6H12. This experiment shows at least that the pro-apoptotic/growth regulatory effects that we report in this study are not simply due to a blocking of cis or trans CD47-SIRPα interactions and are more likely due to agonism of the ED9 antibody that was actually also reported by us before in another context [54].
SIRPα ligation synergizes with conventional antileukemic and targeted agents in t(8;21) and t(15;17) AML cells Considering the potential of exploiting SIRPα targeting to improve the treatment of AML patients, we examined the efficacy of the ED9 mAb in combination with clinically relevant chemotherapeutic agents used for the treatment of AML. NB4 chSIRPα and EV cells were exposed to cytarabine (ARA-C) and daunorubicin (DNR) in combination with ED9 mAb. Survival was monitored after 4 days using a range of chemotherapeutic drug concentrations, which had shown appropriate dose response curves in pilot experiments. We used a fixed concentration of ED9 mAb (10 µg/ml), which had been shown to promote PCD in NB4 cells (Figure 13A). Co-incubation of NB4 cells stably expressing chSIRPα with each of the two chemotherapeutics and ED9 mAb resulted in synergistic effects (combination indexes (CI) using standard calcusyn calculation were CIDNR=0,059±0,04 and
CIARA-C=0,60±0,2). No effect of ED9 mAb was seen in the NB4 EV cells (data not shown). In addition, Kasumi-1 chSIRPα and EV cells were exposed to ARA-C, DNR, and VP16 in combination with ED9. Since the Kasumi-1 cell line has been described to harbor an activating c-kit
69 A Kasumi-1 EV Kasumi-1 chSIRPα
1.3% 95%
SIRPα SIRPα
B control ED9 4.33% 4.44%
EV
11.52% 12.04%
6.17% 11.30% 7-AAD
chSIRPα
9.28% 30.86% Annexin-V
80 Control Irrelevant Ab 60 * ED9
40
Dead cells (%)Dead cells 20
0 EVEV chSIRPSIRP α
C
Time (h) 0 6 24 48 0 24 0 24 -p32
-p17
SIRPα PMN EV
Figure 10. Ligation of chSIRPα induces caspase 3-independent PCD in Kasumi-1 cells. (A) Flow cytometric analysis of SIRPα expression was performed by using ED9 mAb in stable Kasumi-1 cells expressing chSIRPα and EV. (B) Kasumi-1 chSIRPα and EV cells were incubated with 10 µg/ml ED9 mAb and the percentage of cell death was determined after 24 hrs. Annexin V and 7-AAD FACS staining defined that ligation of chSIRPα resulted in increased cell death in chSIRPα Kasumi-1 cells compared to EV control cells. Data are means ± SD calculated from 3 independent experiments using triplicate samples (*: significant difference p<0,05). (C) Kasumi-1 cells expressing chSIRPα or EV were treated with 10 µg/ml ED9 for mentioned time points. Caspase 3 staining shows no cleavage of the p32 subunit. As a positive control for caspase 3 cleavage, human neutrophils (PMN) were incubated at room temperature for 0 and 24 hours.
70 Kasumi-1 EV Kasumi-1ch SIRPα 6 Kasumi-1- ED9 EV 6 Kasumi-1- ED9 SIRP-WT Kasumi-1+ ED9 EV + ED9 2 2 Kasumi-1+ ED9 SIRP-WT + ED9
1 1 3 Number of cells x10 Number of cells x10 p=0.69 p=0.02 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Days Days
Figure 11. SIRPα ligation results in inhibition of proliferation in Kasumi-1 cells. Kasumi-1 cells expressing chSIRPα or EV, were incubated with ED9 mAb for 7 days and cell proliferation was evaluated by daily cell counting. Data are means ± SD calculated from 3 independent experiments using triplicate samples.
mutation [55], we also tested ED9 in combination with the tyrosine kinase inhibitor imatinib mesylate (Figure 13B). Similar to the experiments with the NB4 cell line, this was tested using a 4-day survival assay applying a range of drug concentrations, which had shown appropriate dose response curves in pilot experiments, and a fixed concentration of ED9 mAb (10 µg/ml). The leukemic cell survival of Kasumi-1 EV and chSIRPα cells after incubation with ED9 alone were 98 ± 2,4% and 87 ± 4,4%, respectively. Co-incubation of Kasumi-1 SIRPα cells with each of the four anti-leukemic drugs and ED9 resulted in synergism which was indicated by a shift in the dose-response curve. By using the standard Calcusyn calculations for combination effects, a synergistic effect of ED9 incubation was observed for all applied chemotherapeutic drugs (combination indexes (CI) include: CIARA-C=0,46 ±
0,32; CIDNR=0,74 ± 0,06, CIVP16=0,55 ± 0,066 and CIImatinib=0,75 ± 0,11) (Figure 13B). Taken together, these results show that expression and ligation of chSIRPα provide growth inhibitory effects in t(15;17) and t(8;21) AML cells and this had a synergism with established anti- leukemic drugs.
Discussion Insights into the molecular pathogenesis of AML have paved the way for new treatment strategies that specifically target gene products implicated in induction of the leukemia. In the present study we have investigated the role of SIRPα as a potential target in the treatment of AML. By evaluating SIRPα mRNA levels in both pediatric and adult cohorts of AML patients, we observed a differential expression of SIRPα in AML subtypes. Interestingly, high expression of SIRPα was observed in more mature AML subgroups (M4 and M5) in comparison to immature subtypes, normal bone marrow and CD34+ blast cells. There was interpatient vairability of SIRPα expression between patients with M4 or M5 subtypes, which is probably due to heterogeneity of AML. Nevertheless, higher expression in mature subtypes is consistent with a differentiation related expression of SIRPα. Consistent with this, an upregulation of SIRPα was also observed during differentiation of AML cell lines, which express low endogenous levels of SIRPα.
71 A EV chSIRPα 7,9% 8,4%
control
5% 4,8% 9,5% 9,3% Isotype contol
6,7% 6,4%
8% 21,4% DAPI ED9
I 5,7% 40% 8,6% 9,9%
B6H12
5,9% 5,4%
Annexin-V
B
Figure 12. Blocking anti-CD47 antibody cannot mimic ED9 effects in Kasumi-1 cells. (A) Flow cytometry data of DAPI and Annexin-V staining and (B) Summary graph illustrates the quantified flow cytometric data. Kasumi-1 cells expressing chSIRPα or EV were incubated with ED9 mAb or B6H12 as blocking anti-CD47 antibody. Percentage of cell death was increased significantly in the case of ED9 treatment compared to EV but B6H12 anti-CD47 incubation did not have this effect.
The expression of SIRPα was not correlated to the expression of its ligand CD47, which was ubiquitously expressed on the AML blasts. This is consistent with a recent study performed by Nagahara et al. who showed that increased expression of SIRPα and CD47 was not correlated on breast cancer cell lines. However a stronger correlation was observed in the bone marrow and peripheral blood of breast cancer patients compared to normal cases [45]. They suggest that such host factor characteristics may have implications for prognosis of breast cancer. We also evaluated mRNA levels of other related genes in the various datasets of AML. No clear association was observed between CD47 and SIRPα or different FAB subtypes or karyotypes. In our AML study, the association between SIRPα and prognosis is not strong and does not function
72
A 1.2 Antagonism 1.1 1.0 Additivity 0.9 Syne rgism 0.8 0.7 0.6 0.5 3 0.4
Combination index Combination 0.3 0.2 0.1 0.0 9 ED + C R + ED9 N A- D AR
B 1.2 Antagonism 1.1 1.0 Additivity 0.9 0.8 Synergism 0.7 0.6 0.5 0.4
Combination index Combination 0.3 0.2 0.1 0.0 9 9 D D D9 ED9 + E + + E + E + R ib C A AC DN tin D D VP16 + ED9a ARA-C Im -C +
ARA Figure 13. SIRPα-derived signal synergizes with different antileukemic drugs. Inhibition of cell growth is depicted by combination of ED9 mAb (10 µg/ml) with (A) Ara-C and DNR in NB4 cells expressing chSIRPα (B) Ara-C, DNR, VP16, DAC and imatinib in Kasumi-1 cells expressing chSIRPα. Results are based on means of 3 experiments and are calculated using Calcusyn. as an independent factor. Galbaugh et al showed that SIRPα mRNA was high in triple negative breast cancer and related to an increased invasiveness of the tumor [56]. However, thus far there are no publications that show a clear correlation between SIRPα expression and outcome, even for breast cancer. The lack of differential expression of CD47 in our study might be due to the fact that we investigated the bulk of the AML cells and not the leukemic stem cells, for which it was previously established that CD47expression is associated with poor prognosis [20]. We demonstrate here for the first time, that SIRPα ligation using agonistic monoclonal antibodies inhibits cell growth and promotes cell death induction. Our results suggest that PCD via SIRPα is caspase-independent. This mechanism of cell death induction has been reported previously in the context of AML [57], in which no involvement of caspases in cell death of an AML cell line was observed upon treatment with chemotherapeutic drugs. It should be noted that CD47 ligation
73 also induces caspase-independent PCD [57], and it would seem reasonable to assume that this was due to a potential blocking of cis-interactions between CD47 and SIRPα on AML, rather than to agonistic triggering of both receptors. However, observations show that this was not likely to be the case, since the effects on t(15;17) NB4 cells and t(8;21) Kasumi-1 cells could not be reproduced using blocking antibodies against CD47 (Alvarez et al, not shown). The actual mechanism underlying the pro-apoptotic effect of SIRPα triggering is under investigation. Clearly, one obvious candidate to mediate growth inhibitory signals, particularly in myeloid cells, is the cytosolic tyrosine phosphatase SHP-1. This hematopoietic phosphatase can be recruited and activated upon triggering of SIRPα by its natural ligand CD47, as well as by the agonistic antibody ED9 used herein [54]. It should be emphasized that while the survival and proliferation of AML may not be directly regulated by CD47-SIRPα interactions, there the in vivo life span of leukemic cells may well be affected by them in another way. In particular, recent studies have demonstrated that CD47 can act as an anti-phagocytic or so called “don’t eat me” signal that prevents clearance of human leukemic cells by macrophages in xenogeneic mouse models in vivo [20,21]. Other “don’t eat me” signals such as CD200 have also been shown to be upregulated in multiple tumors including AML [58]. Leukemic stem cells appear to have higher levels of CD47 than normal CD34+ HSC cells and this could provide them with a selective advantage for survival. In this study we show that other signals such as SIRPα can be upregulated to encourage such don’t eat me signals and subsequent evasion from programmed cell removal [19,58]. It must be noted that CD47 signaling can also be regulated through binding to its ligand, thrombospondin-1(TSP-1). This CD47-TSP-1 interaction has been shown to inhibit response to nitric oxide and correspondingly increase radiosensitivity. As a result, blocking such interactions could confer therapeutic radioprotection of normal tissues [59,60,61]. Analysis of AML patients had revealed that higher levels of CD47 are associated with a poor prognosis [17,18]. While the results of these studies indicate that blocking of the interaction between CD47 and SIRPα may be of interest from a therapeutic perspective, our current results suggest that it may, perhaps even simultaneously be beneficial to trigger SIRPα as well. Especially since targeting the CD47–SIRPα phagocytic pathway alone is likely to have toxic effects [58]. In fact, the ED9 antibody against SIRPα that we have used herein appears both capable of triggering programmed cell death as well as to block CD47-SIRPα interaction [62]. Clearly, future studies are needed to generate the suitable agents that can trigger apoptosis via human SIRPα and validate it as a potential treatment target in AML.
Acknowledgement This study was financially supported by Stichting Kinderen Kankervrij (KiKa, Children Cancer Free)
74 References 1. Hann IM, Stevens RF, Goldstone AH, Rees JK, domain-containing protein tyrosine phosphatase Wheatley K, et al. (1997) Randomized comparison substrate 1 regulates the migration of Langerhans of DAT versus ADE as induction chemotherapy in cells from the epidermis to draining lymph nodes. children and younger adults with acute myeloid J Immunol 172: 4091-4099. leukemia. Results of the Medical Research 13. Han X, Sterling H, Chen Y, Saginario C, Brown EJ, Council’s 10th AML trial (MRC AML10). Adult and et al. (2000) CD47, a ligand for the macrophage 3 Childhood Leukaemia Working Parties of the fusion receptor, participates in macrophage Medical Research Council. Blood 89: 2311-2318. multinucleation. J Biol Chem 275: 37984-37992. 2. Goldstone AH, Burnett AK, Wheatley K, Smith AG, 14. Saginario C, Sterling H, Beckers C, Kobayashi R, Hutchinson RM, et al. (2001) Attempts to improve Solimena M, et al. (1998) MFR, a putative receptor treatment outcomes in acute myeloid leukemia mediating the fusion of macrophages. Mol Cell (AML) in older patients: the results of the United Biol 18: 6213-6223. Kingdom Medical Research Council AML11 trial. 15. Jiang P, Lagenaur CF, Narayanan V (1999) Integrin- Blood 98: 1302-1311. associated protein is a ligand for the P84 neural 3. Rowe JM, Neuberg D, Friedenberg W, Bennett adhesion molecule. J Biol Chem 274: 559-562. JM, Paietta E, et al. (2004) A phase 3 study of three 16. Brown EJ, Frazier WA (2001) Integrin-associated induction regimens and of priming with GM-CSF protein (CD47) and its ligands. Trends Cell Biol 11: in older adults with acute myeloid leukemia: a 130-135. trial by the Eastern Cooperative Oncology Group. 17. Barclay AN, Brown MH (2006) The SIRP family Blood 103: 479-485. of receptors and immune regulation. Nat Rev 4. Kaspers GJ, Zwaan CM (2007) Pediatric acute Immunol 6: 457-464. myeloid leukemia: towards high-quality cure of all 18. Tsai RK, Discher DE (2008) Inhibition of “self” patients. Haematologica 92: 1519-1532. engulfment through deactivation of myosin-II at 5. Ravindranath Y (2003) Recent advances in the phagocytic synapse between human cells. J pediatric acute lymphoblastic and myeloid Cell Biol 180: 989-1003. leukemia. Curr Opin Oncol 15: 23-35. 19. Chao MP, Majeti R, Weissman IL (2012) Programmed 6. Hann IM, Webb DK, Gibson BE, Harrison CJ (2004) cell removal: a new obstacle in the road to MRC trials in childhood acute myeloid leukaemia. developing cancer. Nat Rev Cancer 12: 58-67. Ann Hematol 83 Suppl 1: S108-112. 20. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal 7. Martens JH, Stunnenberg HG (2010) The S, et al. (2009) CD47 is an adverse prognostic factor molecular signature of oncofusion proteins in and therapeutic antibody target on human acute acute myeloid leukemia. FEBS Lett 584: 2662-2669. myeloid leukemia stem cells. Cell 138: 286-299. 8. Goyama S, Mulloy JC (2011) Molecular pathogenesis 21. Jaiswal S, Jamieson CH, Pang WW, Park CY, of core binding factor leukemia: current knowledge Chao MP, et al. (2009) CD47 is upregulated on and future prospects. Int J Hematol 94: 126-133. circulating hematopoietic stem cells and leukemia 9. Alcalay M, Meani N, Gelmetti V, Fantozzi A, Fagioli M, cells to avoid phagocytosis. Cell 138: 271-285. et al. (2003) Acute myeloid leukemia fusion proteins 22. Chao MP, Alizadeh AA, Tang C, Jan M, Weissman- deregulate genes involved in stem cell maintenance Tsukamoto R, et al. (2011) Therapeutic antibody and DNA repair. J Clin Invest 112: 1751-1761. targeting of CD47 eliminates human acute 10. Adams S, van der Laan LJ, Vernon-Wilson E, lymphoblastic leukemia. Cancer Res 71: 1374-1384. Renardel de Lavalette C, Dopp EA, et al. (1998) 23. Uno S, Kinoshita Y, Azuma Y, Tsunenari T, Yoshimura Signal-regulatory protein is selectively expressed by Y, et al. (2007) Antitumor activity of a monoclonal myeloid and neuronal cells. J Immunol 161: 1853-1859. antibody against CD47 in xenograft models of 11. Seiffert M, Cant C, Chen Z, Rappold I, Brugger human leukemia. Oncol Rep 17: 1189-1194. W, et al. (1999) Human signal-regulatory protein 24. Chao MP, Alizadeh AA, Tang C, Myklebust JH, is expressed on normal, but not on subsets of Varghese B, et al. (2010) Anti-CD47 antibody leukemic myeloid cells and mediates cellular synergizes with rituximab to promote phagocytosis adhesion involving its counterreceptor CD47. and eradicate non-Hodgkin lymphoma. Cell 142: Blood 94: 3633-3643. 699-713. 12. Fukunaga A, Nagai H, Noguchi T, Okazawa 25. Chan KS, Espinosa I, Chao M, Wong D, Ailles L, et al. H, Matozaki T, et al. (2004) Src homology 2 (2009) Identification, molecular characterization,
75 clinical prognosis, and therapeutic targeting of samples tested for cellular drug resistance using human bladder tumor-initiating cells. Proc Natl the methyl-thiazol-tetrazolium assay. Br J Cancer Acad Sci U S A 106: 14016-14021. 70: 1047-1052. 26. Edris B, Weiskopf K, Volkmer AK, Volkmer JP, 36. Roos D, Voetman AA (1986) Preparation and Willingham SB, et al. (2012) Antibody therapy cryopreservation of cytoplasts from human targeting the CD47 protein is effective in a model phagocytes. Methods Enzymol 132: 250-257. of aggressive metastatic leiomyosarcoma. Proc 37. Heemskerk MH, Hooijberg E, Ruizendaal JJ, van Natl Acad Sci U S A. der Weide MM, Kueter E, et al. (1999) Enrichment 27. Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, of an antigen-specific T cell response by retrovirally Dalerba P, et al. (2012) The CD47-signal regulatory transduced human dendritic cells. Cell Immunol 195: protein alpha (SIRPa) interaction is a therapeutic 10-17. target for human solid tumors. Proc Natl Acad Sci 38. Kinsella TM, Nolan GP (1996) Episomal vectors U S A. rapidly and stably produce high-titer recombinant 28. Seiffert M, Brossart P, Cant C, Cella M, Colonna retrovirus. Hum Gene Ther 7: 1405-1413. M, et al. (2001) Signal-regulatory protein alpha 39. Kaspers GJ, Zwaan CM, Pieters R, Veerman AJ (SIRPalpha) but not SIRPbeta is involved in T-cell (1999) Cellular drug resistance in childhood acute activation, binds to CD47 with high affinity, myeloid leukemia. A mini-review with emphasis on and is expressed on immature CD34(+)CD38(-) cell culture assays. Adv Exp Med Biol 457: 415-421. hematopoietic cells. Blood 97: 2741-2749. 40. Chou TC, Talalay P (1984) Quantitative analysis of 29. Damoiseaux JG, Dopp EA, Neefjes JJ, Beelen RH, dose-effect relationships: the combined effects of Dijkstra CD (1989) Heterogeneity of macrophages multiple drugs or enzyme inhibitors. Adv Enzyme in the rat evidenced by variability in determinants: Regul 22: 27-55. two new anti-rat macrophage antibodies against 41. Hubeek I, Comijn EM, Van der Wilt CL, Merriman a heterodimer of 160 and 95 kd (CD11/CD18). J RL, Padron JM, et al. (2008) CI-994 (N-acetyl- Leukoc Biol 46: 556-564. dinaline) in combination with conventional anti- 30. Hatherley D, Harlos K, Dunlop DC, Stuart DI, Barclay cancer agents is effective against acute myeloid AN (2007) The structure of the macrophage signal leukemia in vitro and in vivo. Oncol Rep 19: 1517-1523. regulatory protein alpha (SIRPalpha) inhibitory 42. Balgobind BV, Van den Heuvel-Eibrink MM, De receptor reveals a binding face reminiscent of that Menezes RX, Reinhardt D, Hollink IH, et al. (2011) used by T cell receptors. J Biol Chem 282: 14567-14575. Evaluation of gene expression signatures predictive 31. Vernon-Wilson EF, Kee WJ, Willis AC, Barclay AN, of cytogenetic and molecular subtypes of pediatric Simmons DL, et al. (2000) CD47 is a ligand for rat acute myeloid leukemia. Haematologica 96: 221-230. macrophage membrane signal regulatory protein 43. Metzeler KH, Hummel M, Bloomfield CD, SIRP (OX41) and human SIRPalpha 1. Eur J Immunol Spiekermann K, Braess J, et al. (2008) An 30: 2130-2137. 86-probe-set gene-expression signature predicts 32. Zhao XW, van Beek EM, Schornagel K, Van der survival in cytogenetically normal acute myeloid Maaden H, Van Houdt M, et al. (2011) CD47-signal leukemia. Blood 112: 4193-4201. regulatory protein-alpha (SIRPalpha) interactions 44. Wouters BJ, Lowenberg B, Erpelinck-Verschueren form a barrier for antibody-mediated tumor cell CA, van Putten WL, Valk PJ, et al. (2009) Double destruction. Proc Natl Acad Sci U S A 108: 18342-18347. CEBPA mutations, but not single CEBPA mutations, 33. Valk PJ, Verhaak RG, Beijen MA, Erpelinck CA, define a subgroup of acute myeloid leukemia Barjesteh van Waalwijk van Doorn-Khosrovani with a distinctive gene expression profile that is S, et al. (2004) Prognostically useful gene- uniquely associated with a favorable outcome. expression profiles in acute myeloid leukemia. N Blood 113: 3088-3091. Engl J Med 350: 1617-1628. 45. Nagahara M, Mimori K, Kataoka A, Ishii H, Tanaka 34. Bennett JM, Catovsky D, Daniel MT, Flandrin G, F, et al. (2011) Correlated expression of CD47 and Galton DA, et al. (1985) Proposed revised criteria SIRPA in bone marrow and in peripheral blood for the classification of acute myeloid leukemia. predicts recurrence in breast cancer patients. Clin A report of the French-American-British Cancer Res 16: 4625-4635. Cooperative Group. Ann Intern Med 103: 620-625. 46. Bullinger L, Dohner K, Bair E, Frohling S, Schlenk 35. Kaspers GJ, Veerman AJ, Pieters R, Broekema RF, et al. (2004) Use of gene-expression profiling GJ, Huismans DR, et al. (1994) Mononuclear cells to identify prognostic subclasses in adult acute contaminating acute lymphoblastic leukaemic myeloid leukemia. N Engl J Med 350: 1605-1616.
76 47. Warrell RP, Jr., de The H, Wang ZY, Degos L (1993) 56. Galbaugh T, Feeney YB, Clevenger CV (2010) Acute promyelocytic leukemia. N Engl J Med 329: Prolactin receptor-integrin cross-talk mediated 177-189. by SIRPalpha in breast cancer cells. Mol Cancer 48. Chomienne C, Ballerini P, Balitrand N, Daniel MT, Res 8: 1413-1424. Fenaux P, et al. (1990) All-trans retinoic acid in 57. Carter BZ, Kornblau SM, Tsao T, Wang RY, Schober acute promyelocytic leukemias. II. In vitro studies: WD, et al. (2003) Caspase-independent cell death structure-function relationship. Blood 76: 1710-1717. in AML: caspase inhibition in vitro with pan-caspase 49. van Beek EM, Cochrane F, Barclay AN, van den inhibitors or in vivo by XIAP or Survivin does not affect 3 Berg TK (2005) Signal regulatory proteins in the cell survival or prognosis. Blood 102: 4179-4186. immune system. J Immunol 175: 7781-7787. 58. Chao MP, Weissman IL, Majeti R (2012) The CD47- 50. Gozzini A, Santini V (2005) Butyrates and decitabine SIRPalpha pathway in cancer immune evasion and cooperate to induce histone acetylation and potential therapeutic implications. Curr Opin granulocytic maturation of t(8;21) acute myeloid Immunol 24: 1-8. leukemia blasts. Ann Hematol 84 Suppl 1: 54-60. 59. Isenberg JS, Maxhimer JB, Hyodo F, Pendrak ML, 51. Liu S, Klisovic RB, Vukosavljevic T, Yu J, Paschka P, et Ridnour LA, et al. (2008) Thrombospondin-1 and al. (2007) Targeting AML1/ETO-histone deacetylase CD47 limit cell and tissue survival of radiation repressor complex: a novel mechanism for valproic injury. Am J Pathol 173: 1100-1112. acid-mediated gene expression and cellular 60. Maxhimer JB, Soto-Pantoja DR, Ridnour LA, Shih differentiation in AML1/ETO-positive acute myeloid HB, Degraff WG, et al. (2009) Radioprotection leukemia cells. J Pharmacol Exp Ther 321: 953-960. in normal tissue and delayed tumor growth by 52. Gottlicher M, Minucci S, Zhu P, Kramer OH, Schimpf blockade of CD47 signaling. Sci Transl Med 1: 3ra7. A, et al. (2001) Valproic acid defines a novel class 61. Soto-Pantoja DR, Miller TW, Pendrak ML, Degraff of HDAC inhibitors inducing differentiation of WG, Sullivan C, et al. (2012) CD47 deficiency transformed cells. Embo J 20: 6969-6978. confers cell and tissue radioprotection by 53. Kircher B, Schumacher P, Petzer A, Hoflehner E, activation of autophagy. Autophagy 8. Haun M, et al. (2009) Anti-leukemic activity of 62. de Vries HE, Hendriks JJ, Honing H, De Lavalette valproic acid and imatinib mesylate on human Ph+ CR, van der Pol SM, et al. (2002) Signal-regulatory ALL and CML cells in vitro. Eur J Haematol 83: 48-56. protein alpha-CD47 interactions are required for 54. Alblas J, Honing H, de Lavalette CR, Brown the transmigration of monocytes across cerebral MH, Dijkstra CD, et al. (2005) Signal regulatory endothelium. J Immunol 168: 5832-5839. protein alpha ligation induces macrophage 63. Olek A, Oswald J, Walter J (1996) A modified and nitric oxide production through JAK/STAT- and improved method for bisulphite based cytosine phosphatidylinositol 3-kinase/Rac1/NAPDH oxidase/ methylation analysis. Nucleic Acids Res 24: 5064- H2O2-dependent pathways. Mol Cell Biol 25: 7181- 5066. 7192. 55. Larizza L, Magnani I, Beghini A (2005) The Kasumi-1 cell line: a t(8;21)-kit mutant model for acute myeloid leukemia. Leuk Lymphoma 46: 247-255.
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The Variola virus CD47 homologue A41L (vCD47) mediates interactions with human SIRP family 4 members on myeloid cells Abstract All known viruses from the Poxviridae family encode a protein with similarity to mammalian CD47, termed vCD47, and the Myxoma virus vCD47 has been shown to be a virulence factor in rabbits. Mammalian CD47 is a ubiquitously expressed transmembrane glycoprotein that functions as a ligand for members of the signal regulatory protein (SIRP) family, including the prototypic inhibitory receptor SIRPα that is selectively expressed on myeloid cells. CD47-SIRPα interactions have been shown to regulate potentially relevant immune effector functions including antibody- dependent cytotoxicity and phagocytosis. We therefore postulated that Variola virus-infected cells express vCD47 on their plasma membrane and that it might interact with SIRPα or other SIRP family members to modulate immune cell function. Here, for the first time, we provide evidence that human SIRP family members interact with the Variola virus vCD47 protein (A41L). We demonstrate that the extracellular Ig-like domain of Variola virus vCD47 binds to human myeloid cells. Furthermore, we show that vCD47 binding is selectively inhibited by antibodies raised against the 2nd extracellular C1-set Ig domain of human SIRPα, which cross-react with other SIRP family members. Conversely, antibodies against the 1st extracellular V-set N-terminal domain of SIRPα that can block endogenous CD47 binding were unable to inhibit vCD47 binding. Intriguingly, ectopic expression and preliminary knock-down experiments suggested that none of the individual SIRP family members were either sufficient or essential for vCD47 binding. Further investigations will be required to identify the molecular basis for vCD47 binding to SIRP family member(s) and the functional consequences(s) that such interactions may have for phagocytes.
Julian Alvarez Zarate1, Karin Schornagel1, Michel van Houdt1, Timo K. van den Berg1
1Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The Netherlands
Manuscript in preparation
80 Introduction Large DNA-containing viruses from the families Poxviridae and Herpesviridae contain, in addition to proteins required for self replication and assembly into virions, genes coding for proteins responsible for manipulating and often evading the immune system of the host (1). Many of these proteins show homology with host proteins, because the genes coding for them have originally been hijacked from the host to be integrated into the virus genome (2). Viral proteins that fall into this category can have several functions to protect the virus against 4 host immunity acting as e.g. virokines, viroceptors, apoptotic inhibitors, and other relevant functions (reviewed in (3)). Clearly, understanding these mechanisms also provides insight into the relevant host immune functions that contribute to the control of viral infection. One example are the viral homologues of CD200, a protein expressed by endothelial and epithelial cells that interacts with the macrophage inhibitory receptor CD200 receptor (CD200R) (4). Viral homologes of this molecule have been found in several members of the Chordopoxvirinae subfamily, including Lumpy skin disease virus (LSDV), Shope fibroma virus (SFV), Myxoma virus (MV), Yaba-like disease virus and Yaba-monkey tumor virus (YMTV). A study by Cameron et al (5) showed that the Myxoma virus CD200 homologue M141R was neither required for infection in a rabbit model per se, nor for virus replication in-vitro. On the other hand its absence strongly decreased the clinical score and mortality rate of the infection, suggesting that it somehow affected virulence. Infections with a Myxoma virus M141R knock-out strain resulted in a higher activation of monocytes/macrophages and lymphocytes. This led them to propose that M141R expressed in infected cells transmits inhibitory signals via CD200R expressed on tissue macrophages and possibly also dendritic cells that would impair host innate and adaptive immune responses. Herpesviridae viruses have also been shown to encode CD200R-like proteins (vCD200R) that interact with CD200 (5). Another example of a Poxviridae gene with putative immune-evasive potential is the viral homologue of CD47 (vCD47). The vCD47 can be found in all characterized poxviruses, including Vaccinia virus and Variola virus (6) (Figure 1). The Vaccinia virus vCD47, also known as the A38L protein, was shown to be expressed on the plasma membrane of infected cells and appeared dispensible for viral replication per se (7). vCD47 of Variola virus, the human poxvirus, also known as A41L, shares only 28% identity to the mammalian CD47, but is nevertheless a clear CD47 homologue because a conserved combination of structural features, which include the single extracellular V-set Ig like domain, the 5 times transmembrane spanning region, and the typical disulfide bond between the Ig domain and one of the extracellular loops of the transmembrane region (7). Actually, the low level of homology between vCD47 and endogenous CD47 is consistent with what has been found for similar viral proteins that have been hijacked from the host, and is probably related to the strong evolutionary pressures associated with host-pathogen interactions (3). Importantly, infection of susceptible rabbits with a knock-out strain of the Myxoma virus demonstrated that vCD47, also known as M128L in Myxoma virus, while being dispensable for viral replication was essential for Myxoma virus virulence. It was also observed that M128L was suppressing the activity macrophages, as indicated by the expression of inducible nitric oxide
81 12 Cowpox virus-V168 --MSRVRISLIYLYT---LVVITTTKTIEYTACNDTIIIPCTIDN----- 40 Ectromelia virus-EVM138 --MLRVRISLIYLYT---LVVITTTKTIEYTACNDTIIIPCTIDN----- 40 Monkeypox virus-A40L --MLRVRILLIYLCT---FVVITSTKTIEYTACNDTIIIPCIIDN----- 40 Variola virus-A41L --MLRVRILLIYLCT---FVVITSTKTIEYTACNDTIIIPCTIDN----- 40 Camelpoxvirus-CMLV158 --MLRVRILLIYLCT---FVVITATKTIEYMACNDTIIIPCTIDN----- 40 Vaccinia virus-A38L --MSRVRISLIYLCT---FMIITSTKTIEYTACNNTIIIPCTIDN----- 40 Homo Sapiens-CD47 MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQN 50 . : * :::. **::*: **:*::*** : * AB Cowpox virus-V168 PTK-YIRWKLDNHDILTYNKTSKTTILSKWHTSARLH-S-LSDSDVSLIM 87 Ectromelia virus-EVM138 PTK-YIRWKLDNHDILTYNKTSKTTILSKWHTSARLH-S-LSDNDVSLIM 87 Monkeypox virus-A40L PTK-YIRWKLDNHDILTYNKTSKTTILSKWHTSARLH-S-LSDSDVSLIM 87 Variola virus-A41L PTK-YIRWKLDNHNILTYNKTSKTIILSKWHTSAKLH-S-LSDNDVSLII 87 Camelpox virus-CMLV158 PTK-YIRWKLDNHDILTYNKTSKTTILSKWHTSAKLH-S-LSDNDVSLII 87 Vaccinia virus-A38L PTK-YIRWKLDNHDILTYNKTSKTTILSKWHTSARLH-S-LSDSDVSLII 87 Homo sapiens-CD47 TTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKM 100 * *::**:..::* *:: : :. :.. .:**::. * * ..*.** : CC’C’’ E 1 Cowpox virus-V168 EYKDILP--GTYTCEDNTGIKS--T-VKLVQRHTNWFNDYQTMLMFIFTG 132 Ectromelia virus-EVM138 EYKDILP--GTYTCGDNTGIKS--T-VKLVQCHTNWFNDYQTTLMFIFTG 132 Monkeypox virus-A40L EYKDILP--GTYTCGDNTGIKS--T-VKLVQRHTNWFNDYQTMLMFIFTG 132 Variola virus-A41L KYKDILP--GTYTCEDNTGIKS--T-VKLVQRHTNWFNDHHTMLMFIFTG 132 Camelpoxvirus-CMLV158 EYKDMLP--GTYTCEDNTGIKS--T-VKLVQRHTNWFNDHHTMLMFIFTG 132 Vaccinia virus-A38L EYKDILP--GTYTCEDNTGIKS--T-VKLVQRHTNWFNDYQTMLMFIFTG 132 Homo sapiens-CD47 DKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPI 150 . .* :. *.*** . :. * ::* ..**. .. *:.**. TM1 F G TM2 Cowpox virus-V168 ITLFLLFLEIAYTSISVVFS---TNLGILQVFGCVIAMVELCGAFLFYPS 179 Ectromelia virus-EVM138 ITLFLLFLEIAYTSISVVFS---TNLGILQVFGCVIAMIELCGAFLFYPS 179 Monkeypox virus-A40L ITLFLLFLEIAYTSISVVFS---TNLGILQVFGCVIAMIELCGAFLFYPS 179 Variola virus-A41L ITLFLLFLEIAYTSISVVFS---TNLGILQVFGCIIAMIELCGAFLFYPS 179 Camelpox virus-CMLV158 ITLFLLFLEIAYTSTSVVFS---TNLGILQVFGCIIAMIELCGAFLFYPS 179 Vaccinia virus-A38L ITLFLLFLEITYTSISVVFS---TNLGILQVFGCVIAMIELCGAFLFYPS 179 Homo sapiens-CD47 FAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPG 200 ::::*:: :: .: . . : * * * :*::: : **:** *. TM3 TM4 Cowpox virus-V168 MFTLRHIIGLLMMTLPSIFLIITKVFSFWLLCKLSCAVHLIIYYQLAGYI 229 Ectromelia virus-EVM138 MFTLRHIIGLLMITLPSIFLIITKVFSFWLLCKLSCAVHLIIYYQLAGYI 229 Monkeypox virus-A40L MFTLRHIIGLLMMTLPSIFLIITKVFSFWLLCKLSCAVHLIIYYQLAGYI 229 Variola virus-A41L MFTLRHIIGLLMMTLPSIFLIITKVFSFWLLCKLSCAVHLIIYYQLAGYI 229 Camelpox virus-CMLV158 MFTLRHIIGLLMMTLPSIFLIITKVFSFWLLCKLSCAVHLIIYYQLAGYI 229 Vaccinia virus-A38L MFTLRHIIGLLMMTLPSIFLIITKVFSFWLLCKLSCAVHLIIYYQLAGYI 229 Homo sapiens-CD47 EYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYI 250 ::*:: ** ::. .: :**: : : * *:. * *: *: .** 2 TM5 Cowpox virus-V168 LTVLGLGLSLKECV--DGTLLLSGLGTIMVSEHFSLLFLVCFPSTQRDYY 277 Ectromelia virus-EVM138 LTVLGLGLSLKECV--DGTLLLSGLGTIMVSEHFSLLFLVYFPSTQRDYY 277 Monkeypox virus-A40L LTVLGLGLSLKECV--DGTLLLSGLGTIMVSEHFGLLFLVCFPSTQRDYY 277 Variola virus-A41L LTVLGLGLSLKECV--DGTLLLSGLGTIMVSEHFSLLFLVCFPSTQRDYY 277 Camelpox virus-CMLV158 LTVLGLGLSLKECV--DGTLLLSGLGTIMVSEHFSLLFLVCFPSTQRDYY 277 Vaccinia virus-A38L LTVLGLGLSLKECV--DGTLLLSGLGTIMVSEHFSLLFLVCFPSTQRDYY 277 Homo sapiens-CD47 LAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ 300 *:*:**.*.: *: .*.**:***. : ::: :.*::: ..*.*:
Cowpox virus-V168 ----- Residues critical for SIRP interaction Ectromelia virus-EVM138 ----- Cysteines participating in disulfide bonds Monkeypox virus-A40L ----- -strands of the Ig-like domain Variola virus-A41L ----- Leader peptide and transmembrane regions Camelpox virus-CMLV158 ----- Residues with potential glycosylation Vaccinia virus-A38L ----- Homo-sapiens-CD47 PPRNN 305
82 Figure 1. Alignment of different viral CD47 proteins to the human CD47.Cowpox virus-V168 (GI:20178537), ▶ Ectromelia virus-EVM138 (GI:22164744), Monkeypox virus-A40L (GI:17975063), Variola virus-A41L (GI: 9627668), Camelpox virus-CMLV158 (GI:18640392), Vaccinia virus-A41L (GI: 137380) and Homo sapiens-CD47 (GI:396705) sequences were obtained from EnsEMBL and National Center for Biotechnology Information databases. Sequences were aligned using the ClustalW2 algorithm.
synthase (iNOS) in lymph node macrophages. Clearly, these findings are consistent with a role for M128L in dampening host immunity, and in particular phagocytes. 4 CD47 was originally described as an integrin binding protein, and termed integrin associated protein (IAP). It was shown to be required for the activation of certain integrins and has been implicated in the regulation of leukocyte transmigration and a variety of other integrin-dependent functions (8-11). The molecule is broadly expressed, on both hematopoietic and non-hematopoietic cells. CD47 also interacts with the inhibitory receptor SIRPα (also known as CD172a, p84, BIT, or SHPS-1), which is primarily expressed on myeloid and neuronal cells (12). CD47-SIRPα interactions regulate, generally in a negative homeostatic fashion, a number of effector functions on phagocytes, including macrophages, granulocytes and dendritic cells. Among these are the clearance of host cells, like red cells and platelets (13;14). In addition, CD47-SIRPα interactions restrict the antibody- dependent phagocytosis and killing of cancer cells (10;15), which frequently over-express the molecule. SIRPα represents the prototypic and only inhibitory member of the SIRP family of paired receptors that contains several members (Figure 2). The term ‘paired’ receptor refers to the presence of members with either inhibitory (i.e. SIRPα) and activating potential (i.e. SIRPβ1 and SIRPβ2). In addition to these there is also SIRPγ, a member without obvious signaling capacity, which is expressed on T cells and NK cells. In humans only SIRPα and SIRPγ (with ~10 fold lower affinity) bind to CD47 (16). Ligands for the activating SIRPβ family members have remained unknown. We hypothesized that in the context of an infection with Variola virus vCD47 may interact with members of the SIRP family. vCD47 expressed on infected cells could for instance suppress immune effector functions by interacting with SIRPα on human phagocytes. In turn, this could be instrumental for the virus to suppress host immunity towards infected cells and as a result to promote virulence. Alternatively, or in addition activating SIRP family members such as SIRPβ1 or SIRPβ2 could act as receptors for virus-infected cells and trigger relevant effector functions. In the present study we have explored these possibilities. Our current findings provide the first evidence for interactions between Variola virus vCD47 and human SIRP family members.
Materials and methods Generation of Fc-fusion proteins and biotinelated swap mutant proteins The extracellular domain (residues 19 to 137) of hCD47 was amplified from the pIAP3 (full length hCD47 in Bluescript from Stratagene) by PCR as described before (17). A synthetic plasmid containing residues 18 to 115 of vCD47 was purchased from ShineGene and used to amplify the extracellular domain of vCD47 (vCD47 EDC). Primers used were EcoR1fw (gag atc gaa ttc aat aga gta cac agc gtg caa tg) and C114rv (gag atc aga tct aaa cca att agt atg acg ttg aac). Amplicons
83 A
V V V
C1 C1 C1 V
C1 C1 C1 V
+ +
Y Y Y Y SIRP SIRP1 SIRP SIRP2
B
SIRPalpha1V QVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVT-TV 59 SIRPalphaBitV QVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVT-TV 59 >74% SIRPbeta1V2 QVIQPDKSISVAAGESATLHCTVTSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVT-TV 59 homology >28% SIRPbeta1V1 QVIQPEKSVSVAAGESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVT-TV 59 SIRPgammaV QMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVT-TV 59 homology SIRPbeta2Cterm WIIQPQELVLGTTGDTVFLNCTVLGDGPPGPIRWFQGAGLSREAIYNFGGISHPKET-AV 59 SIRPbeta2Vterm QVLQPEGPMLVAEGETLLLRCMVVGSCTDGMIKWVKVSTQDQQEIYNFKRGSFPGVMPMI 60 ::**: : : *.: *.* . . * : *.: :: *** .* :
SIRPalpha1 SESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVR----- 110 SIRPalphaBit SDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSV------110 SIRPbeta1V2 SDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDHVEFKSGAGTELSV------110 SIRPbeta1V1 SELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSV------110 SIRPgammaV SDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMAL------110 SIRPbeta2Cterm QAS----NNDFSILLQNVSSEDAGTYYCVKFQRKPNR--QYLSGQGTSLKVKAKSTS 110 SIRPbeta2Vterm QRTSEPLNCDYSIYIHNVTREHTGTYHCVRFD-GLSEHSEMKSDEGTSVLV------110 . * *:** : .:: ..***:**:* : *. **.: :
Residues critical for CD47 interaction Cysteines participating in the intradomain disulfide bond Residues with potential N-glycosylation C
SIRPα1 SIRPαBit SIRPβ1V1 SIRPβ1V2 SIRPβ2Nter SIRPβ2Vter SIRPγ SIRPα1 100 88,18 88,18 91,82 45,45 38,18 74,55 SIRPαBit 100 90 93,64 44,55 40,91 80 SIRPβ1V1 100 90 41,82 40 75,45 SIRPβ1V2 100 42,73 40 80,91 SIRPβ2Nter 100 28,18 41,82 SIRPβ2Vter 100 37,27 SIRPγ 100
Figure 2. The SIRP family. A) Schematic representation of the different members of the human SIRP family. B) Alignment of the V-set Ig-like domains of the different SIRP family members to the CD47-binding V-domain of
SIRPα. SIRPα1 (GI:2052056), SIRPαBit (GI:18426911), SIRPβ1v1 (ENSP00000371018), SIRPβ1v2 (ENSP00000279477), SIRPγ (ENSP00000305529), SIRPβ2 (ENSP00000352849) V N-terminal domain and V C-terminal domain sequences were obtained from EnsEMBL. Sequences were aligned using the ClustalW2 algorithm. C) Amino acid sequence identities among the SIRP family V-set Ig-like domains. Note that based on overall structure and homology SIRPα, SIRPβ1 and SIRPγ form a subfamily among SIRPs in humans (indicated by a dotted line in A).
were digested with EcoRV and BglII and cloned into a predigested pFUSE-hIgG1-Fc1 vector (Invitrogen). Escherichia coli DH5α cells were transformed and grown in LB medium containing Zeocin. Positive clones were selected and grown for further isolation of the plasmid. For the generation of hCD47-Fc and vCD47-Fc fusion proteins CHO Freestyle cells were transfected with the pFUSE hCD47 ECD/vCD47 ECD-IgG1 Fc vectors using 293Fectin as a transfection reagent
84 and cultured in a Freestyle medium (Gibco) supplemented with penicillin-streptomycin at 37°C in an 8% CO2 atmosphere under shaking conditions. 400 ml of supernatant was harvested at day 6 and the Fc fusion proteins were affinity purified using ProteinG-Sepharose beads (Sigma Aldrich) and concentrated using an Amicon-15 Ultra 10kD Millipore system. For the generation of the SIRPαBit and SIRPγ swap mutants plasmids corresponding to each of the biotin tagged mutants (aaa,aag,agg, etc) were transfected into the HEK freestyle system as described above. Purification of the proteins was done using a streptavidin column. 4 Coomassie gel and Western blotting The concentrated Fc-fusion proteins were loaded onto a 12,5% sodium dodecyl sulfate- polyacrylamide gel (SDS-PAGE) and separated under non reducing conditions. The gel was then incubated overnight with Coomassie Brilliant Blue. For Western Blotting lysates of NB4 cells were transferred onto a nitrocellulose membrane (Whatman Schleicher and Schuell), which was then blocked with a 2% milk protein solution in Tris-Buffered Saline and Tween 20 TBST for 1 hour at room temperature. Subsequently, it was incubated first with an anti-SIRPα Ab (AbCam, clone 8120) at 0,5μg/ml or an anti-SIRPβ1 Ab (AbCam, clone B1d5) at 4ug/ml in 2% milk protein/TBST) overnight at 4 °C. After washing the membranes they were incubated with an infrared goat anti-Rabbit IgG (Licor) or an infrared rabbit anti-Mouse (Licor) for 60 min at room temperature. Fluorescence was visualized by an Odyssey Clx infrared imaging system
Cell lines, overexpression and knockdown of SIRP family members CHO and HEK293T cells (obtained from the ATTC) were cultured in IMDM, supplemented with 10% FCS (Bovinco). Freestyle CHO and HEK cells were purchased from Invitrogen and cultured in FreeStyle CHO expression medium (Invitrogen). NB4 cells were cultured in IMDM containing 20% FCS and treated with 1 μM all-trans retinoic acid (ATRA) (Sigma) for 4 to 7 days in the presence of antibiotics (pen/strep) to induce neutrophilic differentiation. Plasmids containing sequences of all SIRP family members were purchased from ShineGene and subcloned into pcDNA3.1+ (Invitrogen) or pIRES puro2 (Addgene). CHO and HEK293t cells were transfected using the method described above for Freestyle 293 cells. Expression levels for each cell line were assessed by FACS and Western blotting. SIRPα and SIRPβ1 were knocked-down in NB4 cells by lentivirus-mediated shRNA constructs from the Mission library (Invitrogen). For SIRPα clone TRCN0000002683 with sequence (CCG GGA GCA TGG TTT CAG GAG TTT ACT CGA GTA AAC TCC TGA AAC CAT GCT CTT TTT) was used and for SIRPβ1 clone TRCN0000029800 with sequence (CCG GTC CTA GTC CTT TCC TGC TGA TCT CGA GAT CAG CAG GAA AGG ACT AGG ATT TTT) was used. Upon transduction with the lentiviral particles the cells were kept under puromycin selection and were regularly checked for expression levels of the targeted protein by flow cytometry and/or Western blotting.
Antibodies, flow cytometry and beads binding assay Monoclonal mouse antibodies 12C4 (mouse IgG1/κ) and 6D6 (mouse IgG2a/κ) were generated as previously described (10). Commercially available antibodies B1D5 (mouse IgG2a Abcam) and
85 C17 (rabbit IgG Santa Cruz) were also used. As a secondary antibodies anti-mouse IgG Alexa 633 (Invitrogen) or anti-rabbit IgG Alexa 633 (Invitrogen) were used in a 1:200 dilution. Cells were incubated for 20 min with indicated antibodies at 10 µg/ml, washed twice and incubated another 20 min with secondary Ab, washed twice and resuspended in 200ul PBS to be measured in a FACS Calibur flow cytometer (BD Biosciences). The hCD47/vCD47-Fc fusion proteins were bound to streptavidin-coupled fluorescent beads of 1 μm diamenter as previously described (10). In brief 20 µl goat-anti human Fc-biotin (0,5 mg/ml Jackson Immuno Research) was added to a mix of 15 µl of streptavidin-coupled beads (Invitrogen) (6,7x106 beads/µl) and 350 µl PBA (PBS with 0,5% BSA and 0,02% azide). This was incubated for 2 hours at 37°C and 600 rpm. Beads were then centrifuged at 14,000 rpm at 4°C for 2 minutes and washed with 500 µl PBA. 0,5 µg of the purified Fc fusion protein in 300 µl PBA was added and this was incubated overnight at 4°C under rotation. The beads were then centrifuged and washed twice with 100 µl PBA. For the binding assay, 5*105 cells were incubated with blocking antibodies (20 μg/ml) or PBS at 37°C. After 30 min 20 µl beads (diluted 1:10 from stock) were added and incubated for another 45 min. Samples were then washed once, resuspended in 200 μl PBS and flow cytometry was performed.
Swap mutants and antibody binding experiments 96 well plates were coated with a 3μg/ml solution containing rat anti mouse light chain kappa monoclonal (Sanquin; MW1443), washed 5 times (PBS/0,02% tween) and incubated with antibodies 6D6 and 12C4 at a concentration of 10μg/ml for 1 hour. After washing, biotinelated swap mutant proteins were incubated for 1hour, washed and the plate was incubated with 1:10000 strep-polyHRP (invitrogen) in PBS/2% milk for 30’. Signal was measures using a plate reader.
Statistics All data are presented as mean plus SEM. Statistical analyses were performed by unpaired Student t-test using Prism software (GraphPad, version 5.01). P values lower than 0,05 were considered significant.
Results To identify possible binding partners for vCD47 we generated chimeric proteins consisting of
the extracellular parts of vCD47 or hCD47 fused to the human IgG1-Fc tail. The recombinant fusion proteins were produced in a mammalian cell system and isolated by affinity purification on proteinG-sepharose columns (Fig. 3). The proteins were coupled to fluorescent beads and cell binding assays were performed as described before for the hCD47-Fc protein (17). Mature myeloid cells, such as macrophages and neutrophils, play an important role in the host defense against poxviral infections (18). A study by Cameron et al. (5) showed that the vCD47 ortholog (M141) encoded by the Myxoma virus and expressed on the surface of infected cells, suppresses macrophage activation upon Myxoma virus infection in rabbits. This suggested a possible ligand and an immune modulatory mechanism for this molecule on myeloid cells.
86 kDa hCD47 vCD47 250
150
100
75 4
Figure 3. Purity of hCD47- and vCD47-IgG1 fusion proteins. Fusion proteins composed of the extracellular Ig-like domains of hCD47 or vCD47 fused to the Fc portion of human IgG1 were produced in freestyle 293 cells, purified on proteinG-sepharose columns. 10ul of each were subjected to SDS-PAGE under non reducing conditions, and stained with Coomassie Brilliant Blue. Concentrations of the purified product were 2,2ug/ul for the hCD47 and 0,3ug/ul for the vCD47. Both proteins have an estimated size of ~75kDa but they migrate at apparent molecular weights of ~ 125 kDa, which is considerably higher and indicates a substantial level of glycosylation (see also Fig. 1).
Preliminary experiments with our Variola virus vCD47 indicated that the molecule was indeed binding to monocytes and neutrophils but not to lymphocytes (unpublished). This possibility was further explored by performing binding assays to ATRA-differentiated human NB4 cells. Indeed, vCD47 protein-coated beads bound to differentiated NB4 cells (Fig. 4A). Binding was highly reproducible, but generally ~2-fold lower than that observed with human CD47-beads, which were used as a positive control in these experiments. Little binding of hCD47 was observed to undifferentiated NB4 cells while vCD47 bound to NB4 cells regardless of differentiation (Fig. 4B). In spite of the relatively low homology of the vCD47 extracellular Ig-like domain to hCD47 and the poor conservation of the amino acid residues in vCD47 that are known to be important for hCD47 binding to SIRPα (Fig. 1) we nevertheless considered that vCD47 could potentially interact with some of the SIRP family members, including perhaps SIRPα, which represents the only inhibitory member of the SIRP family (Fig. 2). In order to test this hypothesis we first measured the expression levels of all SIRPs on ATRA differentiated and undifferentiated NB4 cells (Fig. 4C) to find that all SIRPs are present in these cells and that all except SIRPγ, which is hardly expressed on NB4 cells consistent with its predominant lymphoid expression (ref. 16), are upregulated upon differentiation. Next NB4 cells were preincubated with mAb 12C4 that is known to block interactions between hCD47 and SIRPα, and the binding with vCD47-coated beads was evaluated. As can be seen in Fig. 5A mAb 12C4 completely failed to inhibit vCD47 binding to NB4 cells, while it did effectively block hCD47-SIRPα interactions as already reported before (10). We therefore extended our analysis to a series of other antibodies that had been raised against SIRPα. Interestingly, one of these, the mAb 6D6, which was actually obtained in the same fusion as 12C4 but had not been described before, was found to be a potent inhibitor of vCD47. This antibody recognized both of the polymorphic SIRPα variants, SIRPα1 and SIRPαBIT, that predominate in the Caucasian population as well as SIRPβ1V2while it had little and no reactivity with SIRPβ1V1 and the non-myeloid SIRPγ (Fig. 5B). Using swap mutants of SIRPαBIT and SIRPγ the binding epitope of 6D6 was mapped to the 2nd extracellular C1-set Ig domain of SIRPα
87 (Fig. 5C). In addition, a second mAb B1D5 that was originally raised against SIRPβ1V1 (19), but was
found to cross-react with SIRPαBIT and SIRPγ (Fig. 5B), also inhibited cellular binding of vCD47. Both 6D6 and B1D5 only moderately inhibited binding of hCD47 to NB4 cells. Binding of hCD47 was most
likely mediated by SIRPαBIT since NB4 cells are homozygous for this variant as demonstrated by DNA exon sequencing (data not shown). Furthermore, the mAb OX119, selectively recognizing SIRPγ, which was demonstrated to have a low expression on NB4 cells (Fig. 4C), did neither affect vCD47 nor hCD47 binding (data not shown). Finally, a polyclonal antibody selectively directed against the extracellular domain of the more distantly related activating SIRP family member SIRPβ2 (C17) also inhibited vCD47 binding significantly. Taken together these findings suggested an interaction
A B vCD47 hCD47
hCD47-Fc
ss 34% 48%
C 40 NB4 NB4 ATRA 30 *
20
%Binding cells 10
0 7 4 47 D CD vCD h 8000 NB4 NB4+ATRA 6000
4000 MFI
2000
FACS 0 2 9 7 77 1A 1 6D6 2C4 0 1 C17 1 X O
Figure 4. Human and variola CD47 fusion proteins bind to NB4 cells. A) Esquematic representation of the beads binding assay. B) vCD47 and hCD47 binding to ATRA differentiated NB4 cells. Continuous line represents binding in control conditions, discontinuos line represents binding in the presence of blocking mAb 6D6 in the case of vCD47 and mAb 12C4 in the case of hCD47. C) vCD47 binds to both differentiated and undifferentiated NB4 cells whereas hCD47 binds primarily to ATRA treated NB4 cells. D) NB4 cells with and without ATRA treatment were stained for all SIRP family members with a panel of monoclonal and polyclonal antibodies against SIRPα (mAb 72 or 77) SIRPβ1 (mAb 01A), specific, SIRPγ (mAb OX119), or SIRPβ2 (pAb C17). The specificities of 6D6, B1D5 and 12C4 are shown in Fig.5B.
88 A vCD47 hCD47 20 * 50 * * 40 15 * 30 10 20 %Bindingcells 5 %Bindingcells 10 4 0 0 4 5 l 4 5 ol C 17 ro 6D6 1D C nt 6D6 C17 ontr 12 B o 12C B1D C C B 6D6 B1D5 100000 15000
80000 10000 60000
40000 5000 20000
0 0 1 2 1 IT 2 2 BIT 1 V1 1 V2 B 1 V1 1 V P IRP P R P S IRP P SIRP SI IRP IR IRP SIRP S IRP IRP SIR S S S SIR S S
C17 12C4 15000 50000
40000 10000 30000
20000 5000 10000
0 0 1 IT 1 2 1 IT 1 2 2 B 1 V 1 V2 P B 1 V 1 V P P IR P IR P P P S P P S R SIR SIRP SIR IR IRP SI SIRP SIR SIR S S SIR 12C4 C 6D6 1.0 1.0 ) ) 0.8 0.8
0.6 0.6 of SIRP of SIRP 450 450 0.4 0.4 /A /A 450 450 Relativebinding Relativebinding 0.2 0.2 (A (A
0.0 0.0 a a a g g aa a g gg a g a g a aag gga a gag ggg a gaa aga aag gga a ga ggg
Figure 5. vCD47 and hCD47 bind to neutrophil differentiated NB4 through SIRPs but they use different epitopes. A) Anti SIRP blocking ab’s regulate differentially the binding of vCD47 and hCD47 to NB4 ATRA cells. B) The specificity of all blocking antibodies used was studied by staining a CHO/HEK panel expressing individually each of the SIRPs. C) 6D6 and 12C4 bind different epitopes. Binding experiments using SIRPαBit and SIRPγ swap mutants on an EILISA based binding experiment show differences in the epitope these two antibodies bind.
89 of vCD47 with one or more human SIRP family members. However, the reactivity of most of the antibodies with multiple SIRP family members (Fig. 5B) made it impossible to dissect in this way which of these SIRP family members acted as the main vCD47 binding partner. The different pattern of inhibition with the various antibodies, and particularly the differential effect of mAb 12C4, did nevertheless indicate that if vCD47 was binding to SIRPα it was at least not simply binding to the same epitope as the endogenous hCD47. Next we asked which of the SIRP family members can bind vCD47. To directly address this we individually overexpressed each SIRP family member on CHO or HEK293 cells and performed binding assays with vCD47 and hCD47. hCD47 bound to both SIRPα and SIRPγ expressing CHO cells, as could be expected based on previously reported findings (16), and the interaction was again blocked by incubation with Ab 12C4. In contrary, vCD47 coated beads did not bind to any of the SIRP expressing CHO cells (Fig. 6), suggesting that at least in this cellular context neither of the SIRP family members is sufficient to confer vCD47 binding. Another possible explanation is that a proportion of the SIRP family members forms heterodimeric complexes on the surface and that these act to bind vCD47. In line with this at least SIRPα and SIRPβ1 have each been shown to from homodimers, either in a non-covalent or covalent fashion, respectively (20;21). To test it, we generated co-transfectants expressing SIRPα, β1 and/or β2 and performed binding analyses. As depicted in figure 7 none of the co-transfectants showed binding to vCD47, whereas all of the cells expressing SIRPα bound hCD47. One possible explanation for the fact that none of the individually expressed or co-expressed SIRP family members was able to confer binding is that another factor(s) was required for binding. If so, such factor(s) could either be a cell-type specific posttranslational modification relevant for SIRPs, such as e.g. glycosylation, or an additional protein somehow modifying SIRP conformation. In any case, it would confer binding of vCD47 to NB4 cells, but not to CHO or HEK293 cells. Therefore we used a knock-down approach in NB4 cells to study whether any of the individual
60 hCD47 vCD47 40 hCD47 + 12C4
20
%Binding cells
0 1 2 K IT V1 V2 P E B 1 1 CHO H P IR IR P IRP S S IRP IR S O O S K H K S E CH NB4 ATRA C E H CHO SIRPCHO H
Figure 6. hCD47 but not vCD47 binds to SIRP family members when overexpressed on CHO/HEK cells. SIRPs were individually expressed on CHO/HEK cells and their binding to hCD47- and vCD47-beads was tested. Note that whereas hCD47 binds to both of the SIRPα polymorphic variants and to SIRPγ vCD47 binds to none of the individually expressed SIRP family members. Grey areas on the white bars represents the residual binding upon blocking with the 12C4 mAb. Instead, vCD47 binding to NB4 cells was not affected by 12C4 (see also Fig. 5A).
90 SIRP family members was required for vCD47 binding. First, we verified expression of the individual SIRPs on undifferentiated and differentiated NB4 cells. Reduction of SIRPα levels by 64% (Fig. 8A) resulted in a 67% decrease in binding of hCD47 coated beads while it had no detectable effect on the binding of vCD47 (Fig. 8B). Similarly, a reduction of SIRPβ1 by 85% affected neither hCD47 nor vCD47 binding to NB4 cells significantly. Repeated attempts to knock-down SIRPβ2 failed for technical reasons, unfortunately. While these results did not provide evidence for an involvement of either SIRPα or SIRPβ1, they could clearly not completely exclude such an involvement as well. 4
100 hCD47
vCD47 80
60
40
%Binding cells 20
0 2 1 it 1 i t 4 93T B B 2 , NB 1 V1, 1 1 V2, HEK 1 V 1 V2, HEK 2, , 2, 2 2, HEK HEK EK HEK H
Figure 7.vCD47 doesn’t bind to HEK SIRP cotransfectants. SIRPs were cotransected in different combinations on HEK cells and their binding to hCD47 and vCD47-beads was tested. While binding of hCD47 was observed on all transfectans that expressed SIRPα vCD47 beads only bound to NB4 control cells.
A B SCR KD12
60 SIRP α Scr SIRP KD 40 SIRP1 KD
20 % cells Binding
SIRPβ1 0
47 D47 CD C h v SCR KD10
Figure 8. Reduction SIRPα or SIRPβ expression levels on NB4 cells does not affect vCD47 binding. A) SIRPα and SIRPβ1 expression were reduced on NB4 cells to a 64% and 82% of residual expression respectively as determined by western blotting. B) Decreased expression of SIRPα decreases binding of hCD47 but not that of vCD47. Diminished SIRPβ1expression does not affect binding of either hCD47 or vCD47.
91 Discussion Our results provide the first direct evidence that the Variola virus CD47 homologue vCD47, also known as the A41L protein, interacts with one or more members of the SIRP family on human myeloid cells. Here we show that a fusion protein containing the extracellular domain of the Variola protein A41L binds specifically to NB4 cells. This interaction is blockable by 3 different antibodies raised against SIRPs and we show how this interaction uses a different epitope from the one used by the endogenous CD47. However, in spite of our efforts we have not yet been able to establish the identity of the actual vCD47 binding SIRP family member(s).
Our knock-down experiments, in which SIRPα or SIRPβ1 were knocked down to levels that were substantially below that of normal expression, which at least for SIRPα significantly affected endogenous CD47 binding, did not affect vCD47 at all. Nevertheless these results support the ‘counter-balance theory’ (22;23). This postulates that activating members of paired receptor families may have evolved to counterbalance pathogens that bind to the inhibitory members of the family in order to subvert the immune system and its response to infected cells. This theory was proposed based on several observations. Firstly, several paired receptor families have been shown to have members that bind viral ligands (e.g. NKG2, Ly49, Nkrp1, CD200R, DCIR, CEACAM or PIR) or bacteria (e.g. Siglecs) (23). Secondly, endogenous ligands are known for the inhibitory members of paired receptor families, while the activating family members bind more weakly or not at all to such ligands e.g., SIRP (24) or CD200 (25), and often cells express both inhibitory and activating members of the same paired receptor family. Finally, many of the paired receptor families evolve rapidly, consistent with a strong evolutionary pressure as it can be brought about by infectious diseases (26). In line with this theory we have at least obtained the first indications for a viral ligand binding members of the SIRP family. Clearly, it would make sense from the perspective of the virus to target the inhibitory SIRPα to evade host immunity and the reported requirement of the Myxoma virus vCD47 for establishing a productive infection in rabbits is certainly consistent with this. However, as each host-pathogen relationship may evolve in its own fashion this does not mean that a similar situation applies to the human-variola situation. In this context it is relevant also to consider the inter-species differences in composition of the SIRP family (27). In particular it seems that SIRPα, the only inhibitory member of the SIRP paired receptor family, is strongly conserved among the various mammalian, bird and reptile species in which it has been found. However, among the other family members, which often appear to have activating signaling potential, such as SIRPβ1 or SIRPβ2, the pattern is very different. Here no clear orthologs among species can be identified and it appears that most of these activating SIRPs have arisen independently and relatively recent by duplication and divergence from SIRPα. This is obviously in line with an involvement of SIRP family members in the recognition of vCD47, but it can be anticipated that the situation in this respect could be different for any poxvirus and its specific host. Clearly, more work is needed to establish which of the SIRP family members and potentially other relevant components function in the recognition of Variola virus and other poxviral vCD47 molecules and how this impacts the interplay between virulence and host defense.
92 References 1. Engel, P., and A. Angulo. 2012. Viral 12. Adams, S., L. J. van der Laan, E. Vernon-Wilson, L. C. immunomodulatory proteins: usurping host genes as Renardel de, E. A. Dopp, C. D. Dijkstra, D. L. Simmons, a survival strategy. Adv. Exp. Med. Biol. 738: 256-276. and T. K. van den Berg. 1998. Signal-regulatory 2. Smith, G. L., J. A. Symons, A. Khanna, A. protein is selectively expressed by myeloid and Vanderplasschen, and A. Alcami. 1997. Vaccinia neuronal cells. J. Immunol. 161: 1853-1859. virus immune evasion. Immunol. Rev. 159: 137-154. 13. Oldenborg, P. A. 2013. CD47: A Cell Surface 3. Elde, N. C., and H. S. Malik. 2009. The evolutionary Glycoprotein Which Regulates Multiple Functions conundrum of pathogen mimicry. Nat. Rev. of Hematopoietic Cells in Health and Disease. 4 Microbiol. 7: 787-797. ISRN. Hematol. 2013: 614619. 4. Rygiel, T. P., and L. Meyaard. 2012. CD200R 14. Yamao, T., T. Noguchi, O. Takeuchi, U. Nishiyama, H. signaling in tumor tolerance and inflammation: A Morita, T. Hagiwara, H. Akahori, T. Kato, K. Inagaki, H. tricky balance. Curr. Opin. Immunol. 24: 233-238. Okazawa, Y. Hayashi, T. Matozaki, K. Takeda, S. Akira, 5. Cameron, C. M., J. W. Barrett, L. Liu, A. R. Lucas, and M. Kasuga. 2002. Negative regulation of platelet and G. McFadden. 2005. Myxoma virus M141R clearance and of the macrophage phagocytic expresses a viral CD200 (vOX-2) that is responsible response by the transmembrane glycoprotein SHPS- for down-regulation of macrophage and T-cell 1. J. Biol. Chem. 277: 39833-39839. activation in vivo. J. Virol. 79: 6052-6067. 15. Chao, M. P., A. A. Alizadeh, C. Tang, J. H. 6. Sanderson, C. M., J. E. Parkinson, M. Hollinshead, Myklebust, B. Varghese, S. Gill, M. Jan, A. C. Cha, and G. L. Smith. 1996. Overexpression of the vaccinia C. K. Chan, B. T. Tan, C. Y. Park, F. Zhao, H. E. Kohrt, virus A38L integral membrane protein promotes R. Malumbres, J. Briones, R. D. Gascoyne, I. S. Ca2+ influx into infected cells.J. Virol. 70: 905-914. Lossos, R. Levy, I. L. Weissman, and R. Majeti. 2010. Anti-CD47 antibody synergizes with rituximab 7. Parkinson, J. E., C. M. Sanderson, and G. L. Smith. to promote phagocytosis and eradicate non- 1995. The vaccinia virus A38L gene product is a 33- Hodgkin lymphoma. Cell 142: 699-713. kDa integral membrane glycoprotein. Virology 214: 177-188. 16. Brooke, G., J. D. Holbrook, M. H. Brown, and A. N. Barclay. 2004. Human lymphocytes interact 8. Liu, Y., H. J. Buhring, K. Zen, S. L. Burst, F. J. Schnell, directly with CD47 through a novel member of the I. R. Williams, and C. A. Parkos. 2002. Signal signal regulatory protein (SIRP) family. J. Immunol. regulatory protein (SIRPalpha), a cellular ligand 173: 2562-2570. for CD47, regulates neutrophil transmigration. J. Biol. Chem. 277: 10028-10036. 17. van Beek, E. M., J. A. Zarate, R. van Bruggen, K. Schornagel, A. T. Tool, T. Matozaki, G. Kraal, D. 9. Motegi, S., H. Okazawa, H. Ohnishi, R. Sato, Y. Roos, and T. K. van den Berg. 2012. SIRPalpha Kaneko, H. Kobayashi, K. Tomizawa, T. Ito, N. controls the activity of the phagocyte NADPH Honma, H. J. Buhring, O. Ishikawa, and T. Matozaki. oxidase by restricting the expression of 2003. Role of the CD47-SHPS-1 system in regulation gp91(phox). Cell Rep. 2: 748-755. of cell migration. EMBO J. 22: 2634-2644. 18. Alzhanova, D., and K. Fruh. 2010. Modulation 10. Zhao, X. W., E. M. van Beek, K. Schornagel, H. Van of the host immune response by cowpox virus. der Maaden, M. van Houdt, M. A. Otten, P. Finetti, Microbes. Infect. 12: 900-909. M. van Egmond, T. Matozaki, G. Kraal, D. Birnbaum, E. A. van, T. W. Kuijpers, F. Bertucci, and T. K. van 19. Seiffert, M., C. Cant, Z. Chen, I. Rappold, W. Brugger, den Berg. 2011. CD47-signal regulatory protein- L. Kanz, E. J. Brown, A. Ullrich, and H. J. Buhring. alpha (SIRPalpha) interactions form a barrier for 1999. Human signal-regulatory protein is expressed antibody-mediated tumor cell destruction. Proc. on normal, but not on subsets of leukemic myeloid Natl. Acad. Sci. U. S. A 108: 18342-18347. cells and mediates cellular adhesion involving its counterreceptor CD47. Blood 94: 3633-3643. 11. Kuriyama, T., K. Takenaka, K. Kohno, T. Yamauchi, S. Daitoku, G. Yoshimoto, Y. Kikushige, J. Kishimoto, 20. Liu, Y., I. Soto, Q. Tong, A. Chin, H. J. Buhring, T. Wu, Y. Abe, N. Harada, T. Miyamoto, H. Iwasaki, T. K. Zen, and C. A. Parkos. 2005. SIRPbeta1 is expressed Teshima, and K. Akashi. 2012. Engulfment of as a disulfide-linked homodimer in leukocytes hematopoietic stem cells caused by down- and positively regulates neutrophil transepithelial regulation of CD47 is critical in the pathogenesis migration. J. Biol. Chem. 280: 36132-36140. of hemophagocytic lymphohistiocytosis. Blood 21. Lee, W. Y., D. A. Weber, O. Laur, S. R. Stowell, I. 120: 4058-4067. McCall, R. Andargachew, R. D. Cummings, and
93 C. A. Parkos. 2010. The role of cis dimerization 25. Hatherley, D., and A. N. Barclay. 2004. The CD200 of signal regulatory protein alpha (SIRPalpha) in and CD200 receptor cell surface proteins interact binding to CD47. J. Biol. Chem. 285: 37953-37963. through their N-terminal immunoglobulin-like 22. Hatherley, D., S. C. Graham, J. Turner, K. Harlos, D. domains. Eur. J. Immunol. 34: 1688-1694. I. Stuart, and A. N. Barclay. 2008. Paired receptor 26. Vilches, C., and P. Parham. 2002. KIR: diverse, specificity explained by structures of signal rapidly evolving receptors of innate and adaptive regulatory proteins alone and complexed with immunity. Annu. Rev. Immunol. 20: 217-251. CD47. Mol. Cell 31: 266-277. 27. van Beek, E. M., F. Cochrane, A. N. Barclay, and T. 23. Akkaya, M., and A. N. Barclay. 2012. How K. van den Berg. 2005. Signal regulatory proteins do pathogens drive the evolution of paired in the immune system. J. Immunol. 175: 7781-7787. receptors? Eur. J. Immunol. 24. Barclay, A. N., and M. H. Brown. 2006. The SIRP family of receptors and immune regulation. Nat. Rev. Immunol. 6: 457-464.
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Regulation of phagocyte migration by signal regulatory 5 protein-alpha signaling Abstract Signaling through the inhibitory receptor signal regulatory protein-alpha (SIRPα) controls effector functions in phagocytes. However, there are also indications that interactions between SIRPα and its ligand CD47 are involved in phagocyte transendothelial migration. We have investigated the involvement of SIRPα signaling in phagocyte migration in vitro and in vivo using mice that lack the SIRPα cytoplasmic tail. During thioglycollate-induced peritonitis in SIRPα mutant mice, both neutrophil and macrophage influx were found to occur, but to be significantly delayed. SIRPα signaling appeared to be essential for an optimal transendothelial migration and chemotaxis, and for the amoeboid type of phagocyte migration in 3-dimensional environments. These findings demonstrate, for the first time, that SIRPα signaling can directly control phagocyte migration, and this may contribute to the impaired inflammatory phenotype that has been observed in the absence of SIRPα signaling.
Alvarez Zarate J*,1, Matlung HL*,1, Matozaki T†, Kuijpers TW‡,, Maridonneau-Parini I,§,¶2, van den Berg TK*,2
*Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The Netherlands. †Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan. ‡Emma Children’s Hospital, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands. §CNRS, UMR5089, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France. ¶Université de Toulouse, UPS, IPBS, Toulouse, France.
1, 2 these authors contributed equally
Experimental work was all performed at Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam
Submitted for publication
98 Introduction Phagocytes, including granulocytes and macrophages, play a central role during local inflammation, triggered by infection or by other causes, such as e.g. autoimmunity. Extravasation of phagocytes occurs by a series of highly coordinated events, which consecutively include rolling, firm adhesion, and diapedesis (1). Intercellular adhesion molecules such as selectins, which mediate rolling, and integrins, which are involved in various other steps of the phagocyte transendothelial migration (TEM) process, play a critical role in this, but exactly how the various events are coordinated is not really understood. Once in the tissue phagocytes use chemotactic cues to migrate through the interstitial tissue matrix to the source of infection or inflammation. Interstitial migration can occur by two mechanistically distinct modes. Amoeboid migration 5 represents a form of crawling, which can only occur in relatively loosely organized extracellular matrices, such as fibrillar collagen. It can be performed by essentially all subpopulations of leukocytes, including granulocytes and macrophages, and it depends on activity of the RhoC kinase (ROCK) (2). Mesenchymal migration is a mode of migration in dense extracellular matrices that among leukocytes can only be performed by macrophages (2). It involves proteolytic degradation of the matrix and it is known that specialized adhesion structures termed podosomes play a critical role in this type of migration (3). There are many unanswered questions also regarding the signaling events that coordinate the interstitial migration of phagocytes. Signal regulatory protein alpha (SIRPα), also known as p84, BIT, MFR or SHPS-1, is an inhibitory immunoreceptor selectively expressed in myeloid and neuronal cells (4). Its extracellular domain contains three extracellular immunoglobulin-(Ig)-like domains through which it binds to the broadly expressed CD47 molecule establishing an interaction that can transduce signals downstream of both receptors (5;6). The SIRPα intracellular tail encodes four tyrosine residues that form two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of these ITIM tyrosines, upon CD47 binding or other stimuli, leads to the recruitment and activation of the cytosolic tyrosine phosphatases Src homology region 2 domain-containing phosphatase-1 and/or 2 (SHP-1 and/or SHP-2). In addition to this, SIRPα has been shown to act as a scaffold for a variety of other signaling and adaptor proteins, such as Src kinase-associated phosphoprotein 2 (SKAP2, Adhesion and Degranulation-promoting protein (ADAP) and Protein tyrosine kinase 2 beta (PYK2) (7). Collectively these SIRPα-associated signaling molecules regulate a variety of phagocyte effector functions dedicated to homeostasis and host defense, most often in a negative fashion. Intriguingly, studies in chronic autoimmune inflammatory models performed in SIRPα mutant mice have demonstrated that signaling through SIRPα is indispensable for establishing inflammation and the associated clinical symptoms (8;9). This has mainly been attributed to an essential role for SIRPα signaling in the induction of Th17 responses, which are known to be instrumental in the induction of these different autoimmune inflammatory conditions. In turn this may be linked to a marked decrease in the number of CD11c+ DC in lymphoid tissues of SIRPα-mutant mice (10). However, it seems possible that SIRPα signaling is also acting in a different fashion to affect inflammation, for instance by directly regulating the functions of phagocytes, including neutrophils and macrophages, that are infiltrating the inflamed site. Interestingly, there is in vitro evidence that TEM
99 of neutrophils (11) and monocytes (12) involves interactions between SIRPα and CD47, suggesting a possible direct role of SIRPα in regulating phagocyte extravasation and migration. In the present study we have addressed the role SIRPα signaling in phagocyte migration in vivo. In order to do so we have subjected mice that lack the SIRPα cytoplasmic tail (i.e. SIRPαΔcyt mice) to acute sterile thioglycollate peritonitis. We observed a delayed infiltration of both neutrophils and macrophages into the peritoneum during thioglycolate-induced peritonitis. In addition, we demonstrated that SIRPα signaling is instrumental in TEM. Finally, our in vitro experiments demonstrated that the amoeboid but not the mesenchymal mode of interstitial migration of macrophages was impaired in the absence of SIRPα signaling. Collectively, these findings show, for the first time, that SIRPα signaling can directly control phagocyte migration both in vitro and in vivo, and this contributes to our understanding about the role of SIRPα in immunity and host defense.
Materials and methods Mice and Peritonitis model SIRPαΔcyt mice on a C57Bl6/J background have been described before (13). Where indicated peritonitis was induced by a single i.p. injection of 1 ml of 4% thioglycollate (Sigma, St Louis, MO, USA) in PBS. At the indicated times after injection the mice were sacrificed and the abdominal cavities were flushed with 5 ml ice cold PBS to harvest the cells. All described animal experiments were approved by the Ethical Committee for Animal Experimentation of the Netherlands Cancer Institute in accordance with Dutch law on animal experimentation. All surgery was performed under isoflurane anesthesia and all efforts were made to minimize suffering.
Flow cytometry Extracellular stainings were done to distinguish cell populations from the peritoneum and to determine the levels of integrins on bone marrow derived macrophages (BMDM) and bone marrow derived neutrophils (BMDN). After blocking with anti-CD16/32, cells were stained with Gr1 Ab (clone 1A8), F4/80 Ab, CD3 Ab and B220 Ab to identify populations or with CD11a Ab, CD11b Ab, CD11c Ab, CD18 Ab and CD61 Ab to identify different integrins. All Abs were purchased at eBiosciences (San Diego, CA, USA). Measurements were done in a FACS Calibur flow cytometer (BDbioscience. Bedford, MA, USA).
Isolation and culture of BMDM and BMDN Bone marrow cells were cultured 7 days on complete medium with 20 ng/mL recombinant mouse macrophage colony stimulating factor (rmM-CSF) (eBiosciences Bedford, MA, USA) to obtain BMDM. BMDN were isolated from bone marrow with Gr1 magnetic beads (Miltenyi biotec, Bergisch Gladbach Germany) and incubated overnight with human recombinant granulocyte colony stimulating factor (hrG-CSF) (10ng/ml) and mouse recombinant interferon gamma (mrIFN-γ) (50ng/ml) (Peprotech, London, UK).
100 BMDM and BMDN transwell migration assay Chemotaxis of BMDM and BMDN was assessed by means of Fluoroblock inserts (Falcon, Colorado Springs, CO, USA). 6*105 BMDN or BMDM from WT and SIRPαΔcyt mice were labelled with calcein-AM (Molecular Probes, Invitrogen, Carlsbad, CA, USA) and seeded on the upper chamber of 3μm or 8μm pores respectively. C5a (10nM) was used as a chemoattractant. Cell migration was assessed by measuring fluorescence in the lower compartment at 2’ intervals for 1h (BMDN) or 2h (BMDM) with a HTS7000+plate reader (Perkin Elmer, Waltham, MA, USA) at an excitation wavelength of 485 nm and emission wavelength of 535 nm.
Transendothelial migration assay 5 Brain endothelial (bEnd5) cells (ATCC, Manassas, VA, USA) were seeded in fibronectin coated μ-slides VI 0.4 (Ibidi, Martinsried, Munchen, Germany) until a confluent monolayer was formed and then stimulated with recombinant mouse tumor necrosis factor alpha (rmTNFα) (10ng/ml) (Biovision, Milpitas, CA, USA) for 3 h. BMDN were then flown over the bEnd5 cells at a rate of 90 dyn/cm², left to adhere for 5’, and flown for another 30’. Transmigrating events were monitored by phase contrast with an Axiovert 200 microscope (Zeiss, Jena, Germany)
Immunofluorescence microscopy BMDMs (4 x 104) were seeded on fibronectin-coated Ibidi chambers (μ-Slide VI0,4) overnight. Cells were fixed with paraformaldehyde (3,7%; Sigma St, Louis, MO, USA) for 15’, washed, permeabilized with Triton-X100 (0,1%; Sigma, St Louis, MO, USA) for 10’, washed, and stained with anti-vinculin Ab (clone HVin-1, Sigma, St Louis, MO, USA), followed by FITC-conjugated goat anti–mouse (Invitrogen, Carlsbad , CA, USA) and FITC–coupled phalloidin (Molecular Probes, Invitrogen Carlsbad , CA, USA). Slides were visualized on a Zeiss Jena, Germany) observer microscope.
3D migration assays For Matrigel assays, Matrigel (BD Biosciences, Bedford, MA, USA) was poured in 24 Transwells inserts (8-μm pores Falcon, Colorado Springs CO, USA) and polymerized as described (14). Fibrillar collagen I matrices were prepared by mixing bovine collagen (2 mg/ml) (Nutragen, Advance biomatrix San Diego, CA, USA) and rat tail collagen (4 mg/ml) (BD Biosciences, Bedford, MA, USA). The preparation was added to Transwell inserts (8 μm pores, Falcon, Colorado Springs CO, USA) and allowed to polymerize. Lower and upper chambers of a transwell system (Nunc, Thermo Fisher, Waltham, MA, USA) were filled in with RPMI 1640 containing 10% FCS and 20 ng/mL rmM-CSF (eBiosciences, Bedford, MA, USA) or 1% FCS and 20 ng/mL rmM-CSF, respectively. After starvation for 4 h with RPMI containing 1% FCS BMDMs (2 x 104) cells were seeded in the upper chamber and allowed to migrate. After 48 h z-series images were acquired at 30-μm intervals with a Zeiss (Jena, Germany) observer microscope. The % of migrating cells was determined from z-stack images of the matrix and normalized to the total number of cells in the field of view. Where indicated, the Rho-associated protein kinase (ROCK) inhibitor Y27632 (10 μM) (VWR international, Randor, Pensilvania, USA) was added to the upper and the lower chambers.
101 Gelatin degradation assay Coverslips were coated with 0,2 mg/ml FITC coupled-gelatin (Molecular Probes, Invitrogen, Carlsbad, CA, USA) as previously described (14). BMDMs (1,5 x 105) were cultured for 16h on gelatin-FITC, fixed, processed for phalloidin staining, and observed as described above. Quantification was done by measuring the pixels of total cell surface and the pixels of gelatin- FITC degradation area using Adobe Photoshop software (Adobe Systems, San Jose, CA, USA). Areas of 100 cells were quantified for each condition in 3 separate experiments.
Immunohistochemistry Tissue samples from anterior abdominal wall omentum from WT and SIRPαΔcyt mice were taken, fixed (formalin, 10% Acetic acid), embedded in paraffin, prepared for immunohistochemistry and stained with Gr1-Ab.
Statistical analysis All data are presented as mean plus standard error of the mean (SEM). Statistical analyses were performed by unpaired Student t-test using Prism software (GraphPad, version 5.01). P values lower than 0,05 were considered significant.
Results and Discussion SIRPα signaling supports phagocyte recruitment during acute thioglycolate-induced peritonitis To investigate a role for SIRPα signaling in phagocyte migration in-vivo we subjected mice lacking the SIRPα cytoplasmic tail (designated SIRPαΔcyt) (13) to an acute thioglycollate-induced sterile peritoneal inflammation. Peritoneal lavages were performed to quantify recruitment and determine the composition of the infiltrated cell population. In line with studies previously reported (15;16) phagocyte immigration in wild type (WT) mice occurs in two consecutive waves, which involves first an early influx of predominantly neutrophils peaking at 4 h after thioglycolate injection, followed by an influx of monocytes (which are subsequently defined as macrophages) (Fig. 1). While the overall magnitude of the response did not appear to be substantially altered in SIRPαΔcyt mice, a significant delay in the immigration of both neutrophils as well as macrophages (Fig. 1) was observed. For instance, in SIRPαΔcyt mice the peak of neutrophil infiltration occurred after about 6 h instead of 4 h, as found in WT mice. No differences in the kinetics and magnitude of lymphocyte migration were observed (data not shown), consistent with a phagocyte-selective effect. Furthermore, there were no significant differences in the blood cell counts of neutrophils and monocytes before and during the experiment (Fig. 2A) that could have contributed to the observed delay in phagocyte recruitment and the expression levels of some of the most important integrins for leukocyte extravasation on neutrophils and peripherial blood mononucleated cells (PBMCs) of WT and SIRPαΔcyt showed no difference (Fig. 2B). Of interest, the levels of integrins on neutrophils and mononuclear cells (mainly macrophages) remained similar upon extravasation and infiltration to the peritoneal cavity (Fig. 2C).
102 5
Fig. 1. Delayed recruitment of phagocytes to the peritoneal cavity in SIRPαΔcyt mice. After i.p. injection of thioglycolate into WT and SIRPαΔcyt mice neutrophil and macrophage influx were determined in peritoneal lavages at the indicated time points. Total leukocytes were counted and cell populations were discriminated by FACS. Every time point is representative of at least 3 mice. Asterisk, p≤ 0,05. Note that there is a delay in the migration of both phagocyte populations.
Because SIRPα is primarily expressed on phagocytes (4), including both neutrophils and macrophages, it seemed most likely that the observed supportive role for SIRPα signaling in phagocyte migration was intrinsic to the migrating cells (see also below). However, extrinsic differences, such as e.g. those in the production of chemokines/cytokines, cannot be completely excluded. In this context our preliminary findings upon analysis of peritoneal lavages demonstrated, that there were no notable differences at the early stages of production (i.e. 2 and 6 h) of some of the relevant soluble mediators, such as monocyte chemotactic protein-1 (MCP-1), TNFα and Interleukin-1 beta (IL1β) (data not shown). This is also in line with our previous observation that macrophages from these SIRPαΔcyt animals do not show any differences in cytokine production (7;17). The most important site of leukocyte recruitment during peritonitis in wild type mice is the omentum, and in particular the part associated to the pancreas and stomach (18;19). Immunohistochemical analysis showed that leukocytes were trafficking though the same anatomical areas of the omentum in both SIRPαΔcyt and WT mice (Fig. 2D). Taken together, these findings demonstrate a supportive role for SIRPα signaling in phagocyte migration under acute inflammatory conditions, at least those triggered by thioglycollate intra peritoneal injection, in vivo. Furthermore, they are consistent with a cell- autonomous role for SIRPα signaling in phagocyte migration.
SIRPα∆cyt phagocytes exhibit impaired transendothelial migration and chemotaxis. The migration of leukocytes from the circulation into the peritoneal cavity involves a number of well-defined steps, including extravasation at the postcapillary vessels of the omentum, and the subsequent trafficking through the interstitial tissue towards the peritoneal cavity. In
103 104 ▶ Fig. 2. A) Blood counts of neutrophils and monocytes do not differ between WT and SIRPαΔcyt. After i.p. injection of thioglycolate into WT and SIRPαΔcyt mice neutrophil and monocytes blood counts were determined at the indicated time points. Total leukocytes were counted and cell populations were discriminated by FACS. Every time point is representative of at least 3 mice. B) Neutrophils and PBMC from SIRPαΔcyt mice have similar levels of integrin expression than those of WT. Blood samples were taken at 6h after thioglycollate injection. Blood was lysed and stained for integrins. Neutrophils and PBMC were discriminated based on FSC and SSC. Graphs represent averages ± SEM of at least 3 mice per group. C) Neutrophils and mononuclear cells (MC) from the peritoneal cavity of SIRPαΔcyt mice have similar levels of integrin expression than those of WT. Peritoneal samples were taken at 6h after thioglycollate injection and stained for integrins. Neutrophils and MC were discriminated based on FSC and SSC. Graphs represent averages ± SEM of at least 3 mice per group. D) Neutrophils from SIRPαΔcyt and WT mice extravasate through the stomach and pancreas- associated omentum. Sections from thioglycollate injected mice were prepared for immunohistochemistry and staining with anti-Ly6G. 5
order to study the different aspects of phagocyte migration that are relevant in this context we employed a combination of in vitro migration methods, in which cells from wild type and SIRPαΔcyt mice were compared. First, we studied the capacity of neutrophils and macrophages to perform chemotaxis in a standard transwell assay using complement component 5a (C5a) as the chemoattractant. We observed that both SIRPαΔcyt neutrophils and macrophages transmigrated significantly and consistently less as compared to wild type cells (Fig. 3A). To investigate whether SIRPα signaling also affected phagocyte transmigration in a more physiological context we employed a transendothelial migration assay in which neutrophils migrate across a monolayer of activated mouse bEnd5 endothelial cells. Due to the difficulty in the isolation of sufficient numbers of mouse monocytes from blood this assay could only be performed with neutrophils. Clearly, SIRPαΔcyt neutrophils displayed considerably less transendothelial migration (Fig. 3B), but overall adhesiveness and motility did not appear to be substantially altered (Video 1, see legend for link) Given the critical importance for integrins in the phagocyte transendothelial migration process (1;20), and the reported role of SIRPα signaling in integrin-mediated cytoskeletal arrangement (7), we investigated whether the observed effects could potentially be due to an altered expression of integrins. As can be seen in Fig. 3C there were no significant differences in the levels of expression of many of the relevant integrins. It should be noted that these findings are also consistent with previous observations (7) that demonstrated that SIRPαΔcyt macrophages
do not display differences in the relevant β1,, β2, and β3 integrins. Collectively, these results demonstrate that SIRPα signaling in phagocytes contributes to chemotaxis and transendothelial migration. Clearly, this could, at least in part, provide an explanation for the delayed phagocyte influx into the peritoneal cavity described above.
SIRPα signaling regulates amoeboid but not mesenchymal migration In addition to the above findings, which are primarily relevant in the context of transendothelial migration, we also wanted to explore a potential role of SIRPα signaling in the subsequent 3D migration of phagocytes in interstitial tissues. Basically, there are two modes of leukocyte migration through 3D matrices, designated amoeboid and mesenchymal migration (21). Neutrophils exclusively migrate in the amoeboid mode, whereas macrophages can perform
105 Fig. 3. 2D transwell chemotaxis and transendothelial migration are impaired in SIRPαΔcyt phagocytes. A) C5a-induced 2D migration in transwell chemotaxis assay is regulated by SIRPα signaling. Data shown represents the difference between the maximum and the minimum fluorescent value reached within 1 h in BMDN and 2 h in BMDM. Values shown represent averages ± SEM of n=4 independent experiments. Asterisk, p≤0,05. B) Transendothelial migration is deficient in SIRPαΔcyt BMDN. WT and SIRPαΔcyt BMDN were seeded over a mrTNFα-stimulated monolayer of bEnd5 cells. After 5’ a flow ratio of 0,9dyn/cm2 was applied and transendothelial migration was monitored by time lapse video microscopy using a phase-contrast lens. Data shown represent means ± SEM of 12 measurements done in 3 independent experiments. Asterisk, p≤0,05. C) BMDN and BMDM from SIRPαΔcyt mice have similar levels of integrin expression. Cells were cultured as described in material and methods and stained with specific Abs against the indicated integrins. Gating was based on FCS and SSC. Histograms from representative experiments are shown for BMDN and BMDM. The dotted line represents the isotype control wile the continuous line represents WT (gray filled) or SIRPαΔcyt (no filling) stainings.
either amoeboid or mesenchymal migration depending on the matrix substrate offered (2). Both amoeboid and mesenchymal migration can be studied in in vitro assays using macrophages (2) and we used these to study potential differences between SIRPαΔcyt and WT cells. Furthermore,
106 mesenchymal migration involves the formation of specialized adhesion structures, known as podosomes, which are instrumental in proteolytic degradation of the matrix, a process that can be visualized by plating cells on FITC-labeled gelatin (3). First, we tested the role of SIRPα signaling in mesenchymal migration as assayed by the % of macrophages migrating into 3D matrigel gels within 48 h. As can be seen in Fig. 4A no differences between SIRPαΔcyt and WT macrophages were observed. In line with this, podosome formation (Fig. 4B) and function, as assayed by gelatin degradation (Fig. 4C and 5), were also unaffected by SIRPα mutation, collectively suggesting that mesenchymal migration is normal in SIRPαΔcyt macrophages. In contrary, amoeboid migration in fibrillar collagen I was reduced by more than 50% (Fig. 4D). Parallel experiments with the ROCK inhibitor Y27632 provided further confirmation that both WT as well as SIRPαΔcyt macrophages 5 employed this mode of migration. These findings indicate that SIRPα signaling is selectively regulating macrophage amoeboid migration, but not mesenchymal migration.
Fig. 4. SIRPα signaling in macrophage 3D migration and matrix degradation. A) SIRPα signaling in macrophages does not regulate mesenchymal migration. BMDM were seeded in Matrigel and allowed to migrate for 48 h. Values are averages ± SEM from 3 independent experiments each performed in triplicate. B) Formation of podosomes in BMDM of SIRPαΔcyt mice. BMDM were seeded in fibronectin-coated inserts and were serum starved overnight. Then samples were fixed, permeabilized and stained for F-actin, vinculin and nuclear DNA. Values are averages ± SEM from 3 independent experiments each performed in triplicate. C) SIRPα signaling in macrophage matrix degradation. BMDM were plated in gelatin-FITC coated coverslips and cultured overnight. The samples were then fixed, permeabilized and stained for F-actin and nuclear DNA. The area of degraded gelatin-FITC was quantified and normalized to the area covered by the cells (determined by cortical F-actin staining). Values are averages ± SEM from 3 independent experiments. D) Amoeboid macrophage migration is regulated by SIRPα signaling. Cells were seeded in fibrillar collagen and allowed to migrate for 48 h. To validate the amoeboid mode of migration Rho C kinase inhibitor Y27632 was used at a concentration of 10μM. Values are averages ± SEM from 3 independent experiments each performed in triplicate. Asterisk, p≤ 0,05.
107
Fig 5. Gelatin degradation by WT and SIRPαΔcyt macrophages. BMDM were plated on gelatin-FITC coated coverslips and left for 16h. After fixation samples were stained for phalloidin (red) and DAPI (Blue). Note that the black areas represent the regions of gelatin degradation by BMDM. Left panel shows the BMDM, middle panel depicts gelatin-FITC degradation and right panel are merged images.
Taken together our findings provide direct evidence, for the first time, that SIRPα signaling plays a direct supportive role in phagocyte migration in vitro and in vivo. In addition to the available data demonstrating that TEM of neutrophils and monocytes requires interactions between phagocyte SIRPα and CD47 on endothelial cells (11;12), our detailed in vitro analysis of the different migration properties of SIRPαΔcyt phagocytes provided here, indicates that TEM includes at least one step that is supported by SIRPα signaling, and this could obviously contribute to the delayed phagocyte migration we observed in vivo. Moreover, it could be that the reduced interstitial and in particular the amoeboid-type of macrophage migration through tissues found in SIRPαΔcyt mice may also have contributed to this. Intravital microscopic analysis of phagocyte migration would clearly be necessary to obtain more insight with respect to the relative contributions of these effects. Exactly how the lack of the SIRPα signaling results in these migration deficiencies is not clear yet. Apart from being an inhibitory immunoreceptor that recruits and activates the tyrosine phosphatases SHP-1 and SHP-2, SIRPα has also been shown to be a scaffold protein binding to several intracellular mediators that may transduce signals (22). While effects of SIRPα signaling on migration in murine embryonic fibroblasts were found to be mediated through SHP-2 (13) other players, such as SKAP2 and ADAP, have recently been implicated in regulating cytoskeletal changes downstream of integrins and SIRPα in macrophages (7). In particular, it was shown that integrin- induced actin reorganization was impaired in SIRPαΔcyt or SKAP2-deficient murine macrophages, and a critical role for SKAP2-SIRPα interactions and signaling in this context was also implicated. While amoeboid migration, at least in dendritic cells, appears to be largely integrin independent (23) this does of course not exclude a potential role for SIRPα signaling in macrophage amoeboid migration. Maybe even more importantly, integrins and their downstream signaling and cytoskeletal remodeling are well known to play a prominent role in phagocyte TEM and SKAP2- SIRPα-dependent signals may well be instrumental in this. Nevertheless, further studies will be required to understand the precise role(s) of SIRPα signaling in phagocyte migration.
108 Finally, the role of interactions between SIRPα on phagocytes and CD47 on endothelial cells during TEM needs to be clarified although a number ofin vitro studies suggested that such interactions were required during TEM (11;12). Furthermore, in vivo studies in CD47- deficient mice suggested a role for CD47 inS. aureus-induced peritonitis consistent with the involvement of CD47-SIRPα interactions in phagocyte extravasation. However, more recent studies did not confirm this when normal infiltration of polymorphonuclear cells (PMNs) was observed in CD47-/- mice upon peritoneal insult (24). In the context of in vitro migration into fibrillar collagen I, SIRPα on the migrating macrophages can not be ligated by extracellular CD47 essentially, suggesting regulation of interstitial amoeboid migration by either cis CD47- SIRPα interactions or by ligand-independent SIRPα signaling. 5
Acknowledgements We are gratefull to C. Cougoule and F.X. Frenois for their assistance with histology. Video1. Transendothelial migration of BMDN is regulated by SIRPα signaling. BMDN were flown over a monolayer of endothelial cells for 5 min, allowed to rest on the endothelial cells for other 5 min and then subjected to flow conditions for 20 min. Transmigrating events were recognize as fase-contrast negative BMDN. Link to video: https://www.youtube.com/watch?v=4q74VQB7HEY&feature=youtu.be
References 1. Ley,K., Laudanna,C., Cybulsky,M.I. and mechanisms of the CD47-SIRPalpha signalling Nourshargh,S. (2007) Getting to the site of pathway. Trends Cell Biol., 19, 72-80. inflammation: the leukocyte adhesion cascade 7. Alenghat,F.J., Baca,Q.J., Rubin,N.T., Pao,L.I., updated. Nat. Rev. Immunol., 7, 678-689. Matozaki,T., Lowell,C.A., Golan,D.E., Neel,B.G. 2. van Goethem.E., Poincloux,R., Gauffre,F., and Swanson,K.D. (2012) Macrophages require Maridonneau-Parini,I. and le Cabec, V (2010) Matrix Skap2 and Sirpalpha for integrin-stimulated architecture dictates three-dimensional migration cytoskeletal rearrangement. J. Cell Sci.. modes of human macrophages: differential 8. Okuzawa,C., Kaneko,Y., Murata,Y., Miyake,A., involvement of proteases and podosome-like Saito,Y., Okajo,J., Tomizawa,T., Kaneko,Y., structures. J. Immunol., 184, 1049-1061. Okazawa,H., Ohnishi,H. et al. (2008) Resistance to 3. Linder,S. and Aepfelbacher,M. (2003) Podosomes: collagen-induced arthritis in SHPS-1 mutant mice. adhesion hot-spots of invasive cells. Trends Cell Biochem. Biophys. Res. Commun., 371, 561-566. Biol., 13, 376-385. 9. Tomizawa,T., Kaneko,Y., Kaneko,Y., Saito,Y., 4. Adams,S., van der Laan,L.J., Vernon-Wilson,E., Ohnishi,H., Okajo,J., Okuzawa,C., Ishikawa- Renardel de,L.C., Dopp,E.A., Dijkstra,C.D., Sekigami,T., Murata,Y., Okazawa,H. et al. (2007) Simmons,D.L. and van den Berg,T.K. (1998) Signal- Resistance to experimental autoimmune regulatory protein is selectively expressed by myeloid encephalomyelitis and impaired T cell priming and neuronal cells. J. Immunol., 161, 1853-1859. by dendritic cells in Src homology 2 domain- 5. Isenberg,J.S., Frazier,W.A., Krishna,M.C., Wink,D.A. containing protein tyrosine phosphatase and Roberts,D.D. (2008) Enhancing cardiovascular substrate-1 mutant mice. J. Immunol., 179, 869-877. dynamics by inhibition of thrombospondin-1/CD47 10. Saito,Y., Iwamura,H., Kaneko,T., Ohnishi,H., signaling. Curr. Drug Targets., 9, 833-841. Murata,Y., Okazawa,H., Kanazawa,Y., Sato- 6. Matozaki,T., Murata,Y., Okazawa,H. and Hashimoto,M., Kobayashi,H., Oldenborg,P.A. et Ohnishi,H. (2009) Functions and molecular al. (2010) Regulation by SIRPalpha of dendritic cell
109 homeostasis in lymphoid tissues. Blood, 116, 3517- Roos,D. and van den Berg,T.K. (2012) SIRPalpha 3525. controls the activity of the phagocyte NADPH 11. Liu,Y., Buhring,H.J., Zen,K., Burst,S.L., Schnell,F.J., oxidase by restricting the expression of gp91(phox). Williams,I.R. and Parkos,C.A. (2002) Signal Cell Rep., 2, 748-755. regulatory protein (SIRPalpha), a cellular ligand 18. Cranshaw,M.L. and Leak,L.V. (1990) Milky spots of for CD47, regulates neutrophil transmigration. J. the omentum: a source of peritoneal cells in the Biol. Chem., 277, 10028-10036. normal and stimulated animal. Arch. Histol. Cytol., 12. de Vries,H.E., Hendriks,J.J., Honing,H., De 53 Suppl, 165-177. Lavalette,C.R., van der Pol,S.M., Hooijberg,E., 19. Shah,S., Lowery,E., Braun,R.K., Martin,A., Huang,N., Dijkstra,C.D. and van den Berg,T.K. (2002) Signal- Medina,M., Sethupathi,P., Seki,Y., Takami,M., regulatory protein alpha-CD47 interactions are Byrne,K. et al. (2012) Cellular basis of tissue required for the transmigration of monocytes across regeneration by omentum. PLoS. One., 7, e38368. cerebral endothelium. J. Immunol., 168, 5832-5839. 20. Vestweber,D. (2007) Adhesion and signaling 13. Inagaki,K., Yamao,T., Noguchi,T., Matozaki,T., molecules controlling the transmigration of Fukunaga,K., Takada,T., Hosooka,T., Akira,S. and leukocytes through endothelium. Immunol. Rev., Kasuga,M. (2000) SHPS-1 regulates integrin- 218, 178-196. mediated cytoskeletal reorganization and cell 21. Friedl,P. (2004) Prespecification and plasticity: motility. EMBO J., 19, 6721-6731. shifting mechanisms of cell migration. Curr. Opin. 14. Cougoule,C., Carreno,S., Castandet,J., Labrousse,A., Cell Biol., 16, 14-23. Astarie-Dequeker,C., Poincloux,R., leCabec, V. 22. Timms,J.F., Swanson,K.D., Marie-Cardine,A., and Maridonneau-Parini,I. (2005) Activation of the Raab,M., Rudd,C.E., Schraven,B. and Neel,B.G. lysosome-associated p61Hck isoform triggers the (1999) SHPS-1 is a scaffold for assembling distinct biogenesis of podosomes. Traffic., 6, 682-694. adhesion-regulated multi-protein complexes in 15. Wu,Q., Feng,Y., Yang,Y., Jingliu, Zhou,W., He,P., macrophages. Curr. Biol., 9, 927-930. Zhou,R., Li,X. and Zou,J. (2004) Kinetics of the 23. Lammermann,T., Bader,B.L., Monkley,S.J., Worbs,T., phenotype and function of murine peritoneal Wedlich-Soldner,R., Hirsch,K., Keller,M., Forster,R., macrophages following acute inflammation. Cell Critchley,D.R., Fassler,R. et al. (2008) Rapid leukocyte Mol. Immunol., 1, 57-62. migration by integrin-independent flowing and 16. Call,D.R., Nemzek,J.A., Ebong,S.J., Bolgos,G.L., squeezing. Nature, 453, 51-55. Newcomb,D.E. and Remick,D.G. (2001) Ratio of local 24. Bian,Z., Guo,Y., Luo,Y., Tremblay,A., Zhang,X., to systemic chemokine concentrations regulates Dharma,S., Mishra,A. and Liu,Y. (2013) CD47 deficiency neutrophil recruitment. Am. J. Pathol., 158, 715-721. does not impede polymorphonuclear neutrophil 17. van Beek,E.M., Zarate,J.A., van Bruggen,R., transmigration but attenuates granulopoiesis at the Schornagel,K., Tool,A.T., Matozaki,T., Kraal,G., postacute stage of colitis. J. Immunol., 190, 411-417.
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Disorganized podosomes and impaired adhesion and chemotaxis in macrophages 6 from LADIII patients Abstract Leukocyte adhesion deficiency type 3 syndrome is a rare primary immunodeficiency characterized by an enhanced susceptibility infections and a bleeding disorder. The disease is caused by mutations in the FERMT3 gene resulting in an absence of kindlin3, a protein expressed in hematopoietic cells, including leukocytes and platelets, that binds to integrins and is required for integrin inside-out-activation. Whereas the role of kindlin3 in human neutrophils, lymphocytes and platelets has been described, little is known about the function of the protein in monocytes and/or macrophages. Here, we demonstrate that LADIII patients exhibit a strong in vivo monocytosis, which is associated with defects in macrophage adhesion, spreading and 2D- and 3D- migration. We also provide evidence for a profound disruption of podosome structure and function in LADIII macrophages. These findings demonstrate for the first time a critical role for kindlin3 in macrophage adhesion and migration thereby providing an explanation for the reduction in monocyte extravasation, which may well contribute to the enhanced susceptibility to infection of LADIII patients.
Alvarez Zarate J*,1, Matlung HL*,1, Poincloux R§,¶, Bouissou A§,¶, Kuijpers TW‡,, Maridonneau-Parini I,§,¶2, van den Berg TK*,2
*Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The Netherlands. ‡Emma Children’s Hospital, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands. §CNRS, UMR5089, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France. ¶Université de Toulouse, UPS, IPBS, Toulouse, France. 1 these authors contributed equally 2 these authors contributed equally
Manuscript in preparation
114 Introduction Leukocyte adhesion deficiency type 3 (LADIII) syndrome is a rare autosomal recessive disorder characterized by a bleeding tendency and an enhanced susceptibility to infections (1). The disease is caused by mutations in the FERMT3 gene, which encodes the kindlin3 protein (2;3). Kindlin3 is a hematopoietic protein that binds to the β-chain of integrins, including β1, β2 and β3 and acts as a critical regulator of integrin inside-out activation (4). Integrins play an essential role in cell-cell en cell-matrix interactions and this is important during a variety of processes, including platelet thrombus formation and leukocyte extravasation. During these processes the functional ligand-binding capacity of the relevant integrins is enhanced by a conformational change in the extracellular part of the integrins, which is triggered by extracellular stimuli and their corresponding intracellular signaling pathways, a phenomenon known as integrin inside-out activation (5;6). This critically involves the binding 6 of two proteins, i.e. talin-1 and kindlin3, to the cytoplasmic tail of the integrin β-chain (7). The requirement of kindlin3 for β1 and β3 -integrin function in platelet aggregation that we have previously reported (1;8) explains the bleeding tendency. A recent study with mouse neutrophils suggested that integrin activation occurs by a stepwise process, sequentially involving talin-1 and kindlin3. First, talin-1 is required for inducing conformational extension of the β2 integrin LFA-1, which triggers an intermediate level of affinity, and this conformation mediates the slow rolling process of neutrophils on the endothelial cells of the vessel wall. The subsequent binding of kindlin3 to LFA-1 induces a high-affinity conformation with an arrest of neutrophils on the endothelium (9). The leukocytosis associated with LADIII syndrome illustrates the requirement for kindlin3 for leukocyte extravasation in humans and this also explains the susceptibility to infection and the occurrence of non-pussing infections in particular. However, whereas the functional repercussions of kindlin3 deficiency have been studied extensively in platelets, neutrophils and lymphocytes , the functions of kindlin3 in macrophages have remained largely unknown. This is of particular interest because macrophages, unlike other leukocytes, form specialized adhesion structures, known as podosomes, and exhibit a specialized type of interstitial migration in tissues, known as mesenchymal migration. During this process, podosomes, which contain integrins and most of the F-actin in macrophages, act as sites for docking of lysosomal vesicles and the release of proteolytic enzymes. The latter is important for degradation of the extracellular matrix during mesenchymal migration of macrophages in dense matrices. In the particular case of osteoclasts, which are capable of bone resorption by creating a sealing zone with actin rings, a structure created by the fusion of podosomes on the bone substratum, kindlin3 has shown to be essential for bone resorption. Osteoclasts of kindlin3 deficient mice present podosomes with an altered structure. Schmidt et al. showed that the actin core of podosomes is formed independently of kindlin3 but that formation of the ring around it were integrins, vinculin, paxilin, talin and other podosome markers localize, together with the formation of sealing zones is impaired in the absence of kindlin3 leading to a strong osteopetrosis phenotype in kindlin3-deficient mice (4). A somewhat milder osteopetrotic phenotype is observed in LADIII patients (10-12). All of these phenotypes
115 appear to be the consequence of an inability to properly activate of integrins. Here, we have studied in detail the adhesion and migration bahaviour and podosomes of human macrophages from LADIII patients. This demonstrates for the first time that macrophages from LADIII patients have abnormal podosome organization and display impaired adhesion and chemotaxis.
Material and methods Patient material The study was performed according to national regulations with respect to the use of human materials with written informed consent, and all experiments were approved by the Medical Ethical Committee of the Academic Medical Centre in Amsterdam in accordance with the Declaration of Helsinki. The 8 LADIII patients from which monocytes were isolated for this study are listed in the table below, were the different mutation on the FERMT3 gene are specified. Note that only for Fig.2 the 8 patients were used, the rest of the experiments were performed with patients 1 and 2.
Patient Number Amino-acid or mRNA change Nucleotide change Mutation References 1 p.Asp393ThrfsX29 c.1173delT Deletion unpublished 2 p.Arg509X c.1525C>T Nonsense (1;3;13) 3 p.Arg509X c.1525C>T Nonsense (1;3;13) 4 p.Arg509X c.1525C>T Nonsense (1;3;13) 5 Deletion exon 14 p.Phe558TrpfsX141 c.1671-2A>G Splite site (14) 6 p.Arg573X c.1717C>T Nonsense (3;13;15) 7 p.Arg573X c.1717C>T Nonsense (3;13;15) 8 p.Lys82ThrfsX67 c.238_244dup7 Duplication (16)
Cell counting 250 μl of whole blood was run in an ADVIA (Siemens) to obtain leukocyte differential counts of control and patient material.
Flow cytometry For flow cytometry analysis isolated monocytes were stained with Ab against integrins: CD29-PE (BD Biosciences), CD18-FITC (Pelicluster), CD61-FITC (Pelicluster), β5-PE (eBioscience), CD11a- FITC (eBioscience) and CD11b-FITC (Pelicluster) for 20 min then washed twice and ran on a LSR II cytometer (BD). Results were analyzed with BD FACSDiva Software.
Cell isolation and differentiation Whole blood was separated by percol density gradient as described before (17) and the ring fraction containing the PBMC was harvested and incubated with CD14 Macs Beads (MiltenyBiotech, Auburn,
116 USA) to isolate monocytes according to the procedures recommended by the manufacturer. These monocytes were then cultured with RPMI (Gibco) supplemented with 10% FCS and 20 ng/ ml M-CSF (eBioscience) for 7 to 9 days to differentiate them into macrophages. Macrophages were then harvested by incubation with 10 mM EDTA (Sigma) for 5’ and flushing.
Western blotting Cell lysates of macrophages were prepared by addition of an equal volume of Laemmli sample buffer containing 50 mM DTT and 1%β -mercapto-ethanol and heated for 30’ at 95°C. Samples were put on ice and clarified by centrifugation at 14000 rpm in an Eppendorf centrifuge for 10’ at 4°C. The samples were subjected SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Kindlin3 was detected with antibody T-18 (Santa Cruz Cat-133429). Staining for actin using anti-actin 6 antibody (sc-10731) was performed to control for equal loading. After washing a secondary antibody HRP-coupled anti rabbit (American Biosciences, Buckingham.UK) was used and the signal was detected by enhanced chemiluminescence (Amersham).
Adhesion and spreading assays For the adhesion assay macrophages (4 x 104) were seeded on fibronectin, fibrinogen or vitronectin-coated Ibidi chambers (μ-Slide VI0,4) and left to adhere for 30’ in the presence or absence of RGD and GRGDSP peptides (Bachem 0,5 mM each), then flushed twice and fixed with 3,7% paraformaldehyde (PFA) (Sigma) for 15’. Cells left on the chamber were then counted under an Evos microscope (AMG). For the spreading assay macrophages were seeded on culture plastic or fibrinogen overnight, fixed as described above and stained with phalloidin-FITC. Images were taken with a wide field microscope (Zeiss) and the surface area of approximately 100 cells per condition was determined using Image J software.
Migration assays For 2D migration experiments macrophages were seeded on an 8 μm chip and 2 μl of a 10 nM C5a solution was added as a chemoattractant. Cells migrated over a fibrinogen coated glass and when indicated RGD and GRGDSP peptides were present at a concentration of 5 mM each. The migration of the cells was monitored for 60’, using a time-interval of 30”on a Taxiscan device. Tracks of 20 (LADIII conditions) or 40 (Control conditions) cells were analyzed and velocity and directionality was calculated using the “Chemotaxis and migration tool” plug-in for Image J. Variability of the direction of motion was quantified by the coefficient of variation (CV). For the 3D migration experiments Matrigel (BD Biosciences, Bedford, MA, USA) was poured in 24 Transwell inserts (8 μm pores Falcon, Colorado Springs CO, USA) and polymerized as described (18). Collagen gel matrices were prepared by polymerizing bovine collagen (5 mg/ ml) (Nutragen, Advance biomatrix San Diego, CA, USA) in a solution with HCl 0,01 M and NaOH 0,1 M. Fibrillar collagen I matrices were prepared by mixing bovine collagen (2 mg/ml) and rat tail collagen (4 mg/ml) (BD Biosciences, Bedford, MA, USA). The preparation was added to
117 Transwell inserts (8 μm pores, Falcon, Colorado Springs CO, USA) and allowed to polymerize. Lower and upper chambers of a transwell system (Nunc, Thermo Fisher, Waltham, MA, USA) were filled with RPMI 1640 containing 10% FCS and 20 ng/mL rmM-CSF (eBiosciences, Bedford, MA, USA) or 1% FCS and 20 ng/mL rmM-CSF, respectively. After starvation for 4 h with RPMI containing 1% FCS BMDMs (2 x 104) cells were seeded in the upper chamber and allowed to migrate. After 48 h z-series images were acquired at 30μm intervals with a Zeiss observer microscope. The percentage of migrating cells was determined from z-stack images of the matrix and normalized to the total number of cells in the field of view. Where indicated, the ROCK inhibitor Y27632 (10μM) (VWR international, Randor, Pensilvania, USA) was added to both the upper and the lower chambers.
Immunofluorescence microscopy Macrophages (4 x 104) were seeded on fibronectin-coated Ibidi chambers (μ-Slide VI0,4) overnight. Cells were fixed with 3,7% PFA (Sigma) for 15’, washed, permeabilized with Triton-X100 (0,1%; Sigma, St Louis, MO, USA) for 10’, washed, and stained with anti-vinculin Ab (clone HVin-1, Sigma), followed by FITC-conjugated goat anti–mouse (Invitrogen) and FITC–coupled phalloidin (Molecular Probes, Invitrogen). Slides were visualized on a Zeiss observer microscope.
Proteolytic degradation assay Coverslips were coated with 0,2 mg/ml FITC coupled-gelatin (Molecular Probes, Invitrogen, Carlsbad, CA, USA) as previously described (18). Macrophages (1,5 x 105) were cultured for 16h on gelatin-FITC, fixed with 3,7% PFA (Sigma) for 15’, washed, permeabilized with Triton-X100 0,1% (Sigma) for 10’, washed, and processed for phalloidin staining, and observed as described above. The extent of degradation was assessed by measuring the area of degraded gelatin- FITC on a specific field of view and dividing it by the number of cells within the same image. Quantification was done using Image J. Areas of 100 cells were quantified for each condition in 3 separate experiments.
Lentiviral transduction and live cell imaging Macrophages were transduced with control virus, virus containing a kindlin3-GFP construct (Kindlin3 cDNA was PCR amplified and cloned into pENTR d TOPO (Invitrogen)) and/or LifeAct (19) (Ibidi) on day 4 of differentiation. Lentiviruses were added to the cells in a mix with 0,5 % protamine sulphate, left overnight and then washed twice with PBS. Macrophages were used for experiments on day 7 to 9 of differentiation. For live cell imaging of podosome formation macrophages were serum starved overnight, detached with a solution of 10 mM EDTA (Sigma), plated on glass and immediately visualized using an Axio Observer wildfield fluorescence microscope (Zeiss). The reconstitution spreading experiments were carried out as described above. For the podosome recovery experiment macrophages were plated on fibrinogen, left overnight to form podosomes, fixed, permeabilized and stained for F-actin and vinculin. Approximately 100 cells were counted for each condition.
118 Statistical analysis All data are presented as mean plus SEM. Statistical analyses were performed by unpaired Student t-test using Prism software (GraphPad, version 5.01). P values equal or lower than 0,05 were considered significant.
Results Adhesion and spreading are affected in LADIII macrophages The defects in platelets, neutrophil and lymphocyte adhesion and/or migration of LADIII patients have been characterized in detail, but much less is known about the functioning of monocytes and macrophages from such patients. This is of particular interest since macrophage display unique migration and adhesion behaviour, and form specialized adhesion structures known 6 as podosomes (20). In the present study we evaluated the adhesion and migration phenotype of monocytes and macrophages obtained from two unrelated LADIII patients with distinct mutations (patients 1 and 2 in table in M&M section). One patient, which carries a nucleotide substitution (c.1525C>T) introducing a premature stop codon (p.Arg509X) in exon 12 the FERMT3 gene leading to a complete absence of protein expression in all white blood cell populations has previously been reported (1;3;16;21), whereas the other, which has a nucleotide deletion (c.1173delT) causing a frameshift and a stop codon (p.Asp393ThrfsX29) in exon 9, which also leads to an absence of detectable kindlin3 protein in monocytes (Fig.1). A prominent feature of LADIII patients, which occurs as a result of impaired leukocyte extravasation, is the enhanced number of circulating neutrophils and lymphocytes (16). First, we evaluated whether this was also the case for monocytes by analyzing blood samples from 8 different patients (table in M&M section). Indeed, we observed a profound on average 6-7 fold increase in the number of monocytes circulating in the blood of these patients (Fig 2) consistent with a deficiency in one or more stages of the in vivo extravasation process of these cells. The extent of the monocytosis was comparable to the neutrophilia (Fig. 2) and lymphocytosis (not shown), suggesting that the
LADII Cont
Kindlin3
Actin
Figure 1. Absence of kindlin3 in LADIII macrophages of patient 1. LADIII and control macrophages were lysed and a Western Blot was stained for the presence of Kindlin3. Actin was used as a loading control.
119
Figure 2. Leukocytosis in LADIII syndrome patients. Differential leukocyte counts were obtained from 8 LADIII patients and 5 controls and the neutrophil and monocyte counts are shown. Each symbol represents a separate patient. Note that LADIII patients have a more than 6 fold increase in both circulating neutrophils and monocytes (*: significant differencep <0,05)
defect in extravasation was similar for the different leukocyte populations. In order to evaluate in more detail the adhesion capacity of LADIII macrophages both LADIII and control monocyte- derived macrophages were seeded on different extracellular matrix substrate proteins for 30’. The percentage of adherent cells was markedly reduced in LADIII macrophages (Fig 3a) compared to control cells, demonstrating the kindlin3-dependence of macrophage adhesion to these different substrates. Addition of RGD and GRGDSP peptides further confirmed the integrin dependence of macrophage adhesion. Furthermore, spreading after 7 days of differentiation on plastic, or after overnight culture of mature macrophages on fibrinogen was also dramatically reduced (Fig 3b). Although there is no evidence that the expression of integrins is altered on LADIII leukocytes, including freshly isolated monocytes (1), we wanted to verify this for the relevant integrins on mature monocyte-derived macrophages as well. This was evaluated for the major integrin subtypes expressed by human macrophages, including β1-integrins (CD29), the β2 integrins CD11a/CD18 and CD11b/CD18, and the β3 (CD61) and β5 integrins. We observed a seemingly normal surface expression of all integrin polypeptide chains, with the possible exception of the β3 chain (CD61), which appeared to be reduced to ~40% of normal levels (Fig 4).
Abnormal chemotaxis by LADIII macrophages Neutrophils and lymphocytes of LADIII patients display strong defects in chemotaxis (22;23). However, these leukocyte subsets can only perform the so called amoeboid type of migration, whereas macrophages can perform both amoeboid and mesenchymal migration, depending on the nature of the substrate (24). First, we measured macrophage chemotaxis in real-time with C5a as the chemoattractant on a fibrinogen coated surface. Although the macrophages from both control as well as LADIII patients showed chemotaxis, with comparable levels of directionality (Fig. 5A) and velocity (Fig. 5B) there were very clear differences in the way the cells migrated. Macrophages from healthy donors made long protrusions at the front and the rear of
120 A B
Figure 3. LADIII macrophages exhibit defective adhesion and spreading. A) Adhesion is impaired in LADIII macrophages. Macrophages were plated on different integrin ligand substrates (fibronectin, fibrinogen and 6 vitronectin) and their adhesion was determined. Three fields of view were counted and the average of this is shown for each condition. Where indicated a mix of RGD and GRGDSP peptides (0,5mM each) were added to inhibit integrin-dependent adhesion. B) LADIII spreading is strongly impaired. Macrophages were cultured on plastic or seeded on fibrinogen. The area of more than 100 cells for each condition was measured and expressed as Spreading Index ((Area of spread cell / area of suspension cell)-1). The corresponding p values are 0,0007 and 0,000008 for fibrinogen and plastic respectively, results represent the averages of 3 different independent experiments performed in duplicate. Relative expression
5 b 11 D CD29 CD18 CD61 CD11a C
Figure 4. Integrin expression on LADIII macrophages. Macrophages from the two LADIII patients were stained for the macrophage relevant integrins, results are depicted in a bar graph normalized to their respective controls with means and SD. Results are representative of 2 independent experiments.
the cells. In contrast, macrophages from LADIII patients made a large and wide leading edge, with almost no formation of a rear tail at all (Fig 5C and D). Another striking observation was that during migration the leading edge of a considerable proportion of the LADIII cells (~25%) would tear off the cell body. To our complete surprise these leading edge fragments would continue their directed migration independently of the cell body (Fig. 5E, F and Video1, see link on legend). This was a unique phenomenon that was never observed by us before with any of the control cell preparations (n>8) evaluated. To show that the migration is integrin-dependent we blocked integrin adhesion by adding RGD and GRGDSP peptides (Fig 5). Of interest in the
121 presence of RGD and GRGDSP peptides control cells, but not the LADIII macrophages, migrated considerably faster, this is in line with the idea that extensive integrin activation can inhibit migration (25). In addition some events of leading edge rupture were also observed on control cells migrating in the presence of RGD and GRGDSP peptides (Video 1, see link on legend). The lack of enhanced migration triggered by RGD and GRGDSP in LADIII patients clearly suggests that this is a kindlin3-dependent process. By comparison to macrophages, when neutrophils of LADIII patients were tested in the real-time chemotaxis assay using C5a as the chemoattractant, chemotaxis appeared to be substantially affected (Fig 5G), whereas the morphology of the cells was not different from that of control cells. Furthermore, no leading edge breakage was seen in neutrophils from LADIII patients. Thus, despite their defect in cell adhesion and spreading (Fig. 3), and in contrary to neutrophils from LADIII patients, macrophages from the same patients do not exhibit defective chemotaxis with respect to the speed or directionality of their migration. Thus, LADIII macrophages display unique defects in their migration behaviour and morphology, which includes the lack of rear tail formation and the shedding of large cellular fragments that maintain directional migration capacity.
Kindlin3 regulates podosome structure and dynamics Podosomes are the main specialized adhesion structure associated with integrins in macrophages, and it is via these structures that macrophages can release proteolytic enzymes in a directional fashion, which in turn is important for e.g. the degradation of dense extracellular matrixes during interstitial migration in tissues (26). Podosome formation was evaluated upon adhesion of control or LADIII macrophages to fibrinogen. Typical podosomes on normal macrophages are characterized by an F-actin core surrounded by various proteins including vinculin, talin, CD44, β1 and β2-integrins and cortactin (27;28) (Fig 6A). LADIII macrophages, on the other hand, formed clusters of very unusual, large and circular adhesion structures (Fig 6). F-actin was strongly enriched in these structures, with most intense expression on the edges (Fig. 6). Several protein markers of podosomes (i.e. vinculin, CD44, β1 and β2-integrins and cortactin) were also strongly accumulating in these structures (Fig 6B) but although podosomes from both control and LADIII cells shared all the
Figure 5. Chemotactic properties of LADIII macrophages. C5a-induced chemotaxis by LADIII macrophages on fibrinogen is characterized by normal performance in terms of velocity or directionality, but with striking morphological abnormalities. A) LADIII macrophages migrate as fast as control cells. B) LADIII macrophages exhibit normal directionality during chemotaxis. The variability on the direction of migration is expressed by the coefficient of variation (CV). C, D) Rear tail formation is dependent on integrin activation by kindlin3. Whereas control cells commonly formed rear end tails during chemotaxis (indicated with arrows), these were absent for LADIII macrophages. E, F) Shedding of cytoplasmic fragments of the leading edge of LADIII cells during chemotaxis. Note that the shed cytoplasmic fragments continue directional migration, leaving the immobile cell body behind. G) LADIII neutrophils show impaired chemotaxis. Methods for determining accumulated distance, directionality and speed are described in the M&M section. Results are representative of 2 independent experiments performed in 2 different patients in duplo. In A,B and G individual cells were considered independent events so statistics could be performed. ▶
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123 typical podosome markers, their molecular architecture was clearly very disturbed and the canonical structure of an F-actin core surrounded by the above mentioned markers was absent. Despite the disorganized nature of these podosome–like structures, they did act as adhesion structures and were the only parts of the cells that were actually anchored to the substrate ( Fig. 6 and Video 2, see link in legend). In order to investigate whether the observed abnormal podosome-like adhesion structures of LADIII macrophages nevertheless actually behave like podosomes with respect to the capacity to secrete proteolytic enzymes (24), we next plated macrophages from the LADIII patients and control cells on gelatin-FITC coated surfaces and quantified their gelatin degradation overnight (Fig 7). Degradation by LADIII cells was impaired by more than 50% in total on a single cell basis as compared to control macrophages (Fig 7B), but the LADIII may cover less surface as a consequence of their reduced spreading (see Fig. 3B). A
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Figure 6. Kindlin3 regulates podosome structure and dynamics. Macrophages from control and LADIII patients were plated on fibrinogen, fixed, permeabilized and stained for the indicated podosome markers A) Comparison of control and LADIII podosomes. B) LADIII podosomes express canonical markers but have impaired structure. Scales bars on all images represent 5μm.
124 careful analysis of the degradation by these LADIII cells showed that the degradation observed was exclusively associated with the podosome-like structures structures (Fig 7A). However, whereas normal podosomes have a highly dynamic nature, the podosome-like structures of LADIII macrophages appeared to form very stable adhesion structures on the substrate, which restricted cellular mobility. In order to investigate the dynamics of the podosomes of LADIII macrophages these cells were transduced with a LifeAct-mCherry lentivirus to visualize F-actin. Live cell imaging showed that when LADIII macrophages were spreading on glass coverslips, they rapidly formed these circular disorganized podosome-related structures (Video 2 and data not shown). Moreover, the structures remain remarkably stable over time showing a lifespan that exceeded several hours. In contrast, normal podosomes are very dynamic structures with an average lifespan of about 10 min (24;26). Therefore the decreased gelatin degradation observed, which was quantified as average surface of gelatin degradation per cell, might not so much have been the result of an intrinsic difference in the release of proteolytic enzymes, 6 but rather of a reduced dynamics of podosomes altogether, allowing the cell to cover and also degrade much less surface area of the substrate. Thus, kindlin3 deficiency leads to the formation of very stable and disorganized, but nevertheless functional podosomes.
LADIII macrophage migration in 3D matrices is not affected In contrary to other hematopoietic cells, including neutrophils and lymphocytes, which can only perform the amoeboid type of migration, macrophages can actually use two modes for
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Figure 7. LADIII macrophages exhibit normal podosome function. A) Image of FITC labeled gelatin degraded by control and LADIII macrophages. Red is F-actin, green is Gelatin and blue DAPI. Although the overall degradation is reduced on the LADIII cells a careful look reveals degradation correlating to the podosome like structures of the LADIII macrophages. B) Quantification of gelatin degradation per cell. Results are representative of 2 different independent experiments performed in triplicate.
125 interstitial migration in tissues. In relatively open porous matrices, they use the ‘amoeboid’ mode of migration and in dense poorly porous matrices, they use ‘mesenchymal’ migration. As indicated before the latter involves proteases secreted at podosomes essentially to create paths through the matrix. In fibrillar collagen I, a matrix forming large pores (>2 µm), LADIII macrophages exhibited the typical round and blebbing cell shape of the amoeboid movement that control cells demonstrate (Fig 8A) and the percentage of cells infiltrating the matrix and the migration distance after 48h were comparable for LADIII and control macrophages (Fig 8B). When seeded on dense matrices, i.e. matrigel or gelled collagen I, LADIII macrophages, which have an adhesion defect as shown before (Fig. 3), rolled to the lower point of the meniscus formed during the polymerization process, which precluded a proper quantification of the total cell number and therefore also accurate determination of the proportion of migrated cells. However, we were able measure the distance covered by the migrating macrophages into the matrices after 48h of migration, and this was shown to be comparable for control and LADIII macrophages in both types of matrices. Furthermore, macrophages of both control cells and LADIII patient cells exhibited the characteristic elongated cell shape of the mesenchymal movement Fig 8A. In conclusion, macrophages from LADIII patients do not exhibit any obvious 3D migration defect in either the mesenchymal or amoeboid mode.
Kindlin3 reconstitution recovers spreading and podosome formation in LADIII macrophages To formally prove that the spreading deficiency and the podosome structure and dynamics phenotype observed on the LADIII macrophages was a direct consequence of the absence of kindlin3 we performed reconstitution experiments. LADIII macrophages were transduced with kindlin3-GFP. GFP-positive macrophages were evaluated for their spreading and podosome formation capacity. As depicted in Fig 9, cells transduced with the kindlin3-GFP construct were able to spread as well as control cells (Fig. 9B), lost the ability to form the type of aberrant podosomes described above, and at least partially recovered their ability to form normal podosomes (Fig 9C). These finding demonstrate that the observed features are primarily a direct consequence of the absence of kindlin3 in macrophages.
Discussion Available evidence suggests that kindlin3 plays a critical role in both the adhesion and migration of leukocytes. Kindlin3 is known to bind to at least several of the integrin β-chains, including β1, β2 and β3 integrins, and is believed to play a critical role in the inside-out activation of these integrins (9;11). More recent findings indicate that there may actually be multiple stages of integrin activation, at least in hematopoietic cells, and this involves the coordinated actions of talin1 and kindlin3. Particularly, talin1 appears essential for establishing an intermediate stage of integrin activation, whereas kindlin3 is required for the subsequent full activation (9). Here we report that in macrophages of LADIII patients that lack kindlin3 there is clearly impaired adhesion and spreading, which is consistent with that seen in other leukocytes (29;30). However, the overall performance
126 A Collagen Gel Collagen Fibrilar
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Figure 8. LADIII macrophage migration in 3D matrices is normal. A) Photomicrographs of control and LADIII macrophages embedded in fibrillar or gel collagen. No differences are noted between LADIII and control cells when they adopt amoeboid or mesenchymal shapes in fibrillar or gelled collagen, respectively. B) 3D migration of macrophages is kindlin3 independent. In both porous and non porous matrices the LADIII macrophages perform similarly as control cells. Results are representative of 2 (Collagen fibrillar), 3 (Matrigel) and 1 (Collagen gel) independent experiments performed in triplicate.
127
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Figure 9. Kindlin3 reconstitution in LADIII macrophages recovers spreading and podosome formation. A) Kindlin3 transfected cells were able to spread normally and podosome formation occurs. Control and LADIII cells were transduced with control-mCherry or Kindlin 3-mCherry vectors and analyzed under the microscope. B) LADIII cells transfected with kindlin3 recover from their spreading-deficiency. The spreading index of >50 cells was determined per condition and the averages ± SEM are shown. C) Kindlin3 transfection on LADIII podosomes partially recovers podosome formation. Again over 50 cells per condition were evaluated for the presence of podosomes and the percentage of podosome-containing cells is shown. Results are representative of 1 experiment performed in triplicate.
128 of macrophages in different migration assays, including either chemotaxis on 2D substrates or amoeboid or mesenchymal migration in 3D matrices, was actually not substantially affected. The latter is of interest given the fact that previous studies have shown a strong integrin dependency for macrophage chemotaxis (31;32) This is also in line with our own findings which show that the integrin-inhibitory RGD and GRGDSP peptides are able to inhibit the chemotaxis of LADIII macrophages, which provides direct evidence for integrin involvement at least in the chemotaxis assay that we have used in the present study. Therefore, it appears that kindlin3 is not required for all integrin-dependent processes in macrophages. Of interest, previous studies on macrophages have suggested that there is a delicate interplay between adhesion strength and migration ability with an optimal point for intermediate adhesion (25). Our results are consistent with the idea that an intermediate level of integrin activation in LADIII macrophages is sufficient to perform as good as control macrophages. It should be noted in this context that in the case of neutrophils adhesion, 6 spreading as well as chemotaxis, including that induced by C5a (Fig 5G and (23)), these are clearly all dependent on the presence of kindlin3. Therefore it could be that full integrin activation is less important in the migration of macrophages as compared to neutrophils, or that in macrophages the broad range of integrins expressed makes it possible for putative kindlin3-independent integrins to substitute for the absence of this protein. One possible reason why neutrophil accumulation in tissues might be subject to additional control mechanisms, including a requirement for kindlin3, could be related to their higher potential capacity for tissue damage. In spite of this difference it appears that the overall defect in the extravasation of monocytes and neutrophils is comparable. This may primarily be explained by the reduced adhesive capacity of both cell types, as integrin-dependent firm adhesion is an integral and essential part of the leukocyte extravasation process (33). Besides a normal overall performance of LADIII macrophages in chemotaxis experiments it was clear from our real-time analysis that LADIII macrophages migration is very abnormal. First, they form little or no rear tails, a process that is known to be dependent on integrins in neutrophils, eosinophils (34) and fibroblasts (35) Secondly, they showed a striking shedding of large cytoplasmic fragments from the leading edge. Perhaps even more surprisingly the shed fragments maintain directional chemotaxis behaviour, a phenomenon that has according to our knowledge not been observed before. The precise underlying mechanisms have still to be discovered, but our results support the idea that kindlin3 plays a vital role in the coordination of integrin activation and deactivation. Another prominent feature we observed was that LADIII macrophages formed disorganized podosome-like structures. These podosomes did not only harbor many of the characteristic podosome proteins, albeit in a disorganized fashion, but also retained the ability for proteolytic degradation. Clearly, these LADIII podosomes were found to be much less dynamic than normal podosomes. Nevertheless, the mesenchymal migration that is believed to involve macrophage podosome formation and function appeared essentially unaffected. Clearly, these findings suggest that there is a considerable level of flexibility of podosome structure and dynamics relative to the functional aspects (i.e. proteolytic protein secretion and mesenchymal migration), and this raises questions about the exact structure-function relationship of podosomes.
129 Taken together, these studies show that macrophages of LADIII patients have an impaired adhesion and spreading, which likewise explains their reduced extravasation and thereby their accumulation in the circulation. LADIII macrophages also display abnormalities in podosome formation and chemotaxis that may further hamper their interstitial migratory capacity.
Video 1. Control and LADIII macrophages migrating on a C5a gradient. The leading fragment of some LADIII macrophages breaks loose from the cell body and continues to migrate. Note the different morphologies adopted by the macrophages on the different conditions. Link to the video: https://www.youtube.com/watch?v=oGfHE39N1Y0&feature=youtu.be
Video 2. Disturbed Podosome dynamics on LADIII macrophages. Wile control podosomes are rapidly changing location and present a very dynamic nature the actin cores of the podosome- like structures on LADIII macrophages stay fixed for hours. The fluorescent signal corresponds to F-actin. Link to the video: https://www.youtube.com/watch?v=kzwCVFUYZbQ&feature=youtu.be
References 1. Kuijpers,T.W., Van Lier,R.A., Hamann,D., de signaling in integrin-mediated cell adhesion, Boer,M., Thung,L.Y., Weening,R.S., Verhoeven,A.J. spreading, and retraction. Curr. Opin. Cell Biol., and Roos,D. (1997) Leukocyte adhesion deficiency 24, 600-606. type 1 (LAD-1)/variant. A novel immunodeficiency 8. van de Vijver., De Cuyper,I.M., Gerrits,A.J., syndrome characterized by dysfunctional beta2 Verhoeven,A.J., Seeger,K., Gutierrez,L., van integrins. J. Clin. Invest, 100, 1725-1733. den Berg,T.K. and Kuijpers,T.W. (2012) Defects in 2. McDowall,A., Svensson,L., Stanley,P., Patzak,I., Glanzmann thrombasthenia and LAD-III (LAD-1/v) Chakravarty,P., Howarth,K., Sabnis,H., Briones,M. syndrome: the role of integrin beta1 and beta3 in and Hogg,N. (2010) Two mutations in the platelet adhesion to collagen. Blood, 119, 583-586. KINDLIN3 gene of a new leukocyte adhesion 9. Lefort,C.T., Rossaint,J., Moser,M., Petrich,B.G., deficiency III patient reveal distinct effects on Zarbock,A., Monkley,S.J., Critchley,D.R., leukocyte function in vitro. Blood, 115, 4834-4842. Ginsberg,M.H., Fassler,R. and Ley,K. (2012) Distinct 3. Kuijpers,T.W., van de Vijver,E., Weterman,M.A., roles for talin-1 and kindlin-3 in LFA-1 extension and de,B.M., Tool,A.T., van den Berg,T.K., Moser,M., affinity regulation. Blood, 119, 4275-4282. Jakobs,M.E., Seeger,K., Sanal,O. et al. (2009) 10. Mory,A., Feigelson,S.W., Yarali,N., Kilic,S.S., LAD-1/variant syndrome is caused by mutations in Bayhan,G.I., Gershoni-Baruch,R., Etzioni,A. and FERMT3. Blood, 113, 4740-4746. Alon,R. (2008) Kindlin-3: a new gene involved in 4. Schmidt,S., Nakchbandi,I., Ruppert,R., Kawelke,N., the pathogenesis of LAD-III. Blood, 112, 2591. Hess,M.W., Pfaller,K., Jurdic,P., Fassler,R. and 11. Malinin,N.L., Zhang,L., Choi,J., Ciocea,A., Moser,M. (2011) Kindlin-3-mediated signaling from Razorenova,O., Ma,Y.Q., Podrez,E.A., Tosi,M., multiple integrin classes is required for osteoclast- Lennon,D.P., Caplan,A.I. et al. (2009) A point mediated bone resorption. J. Cell Biol., 192, 883-897. mutation in KINDLIN3 ablates activation of three 5. Etzioni,A. and Alon,R. (2004) Leukocyte adhesion integrin subfamilies in humans. Nat. Med., 15, 313-318. deficiency III: a group of integrin activation 12. Sabnis,H., Kirpalani,A., Horan,J., McDowall,A., defects in hematopoietic lineage cells. Curr. Opin. Svensson,L., Cooley,A., Merck,T., Jobe,S., Allergy Clin. Immunol., 4, 485-490. Hogg,N. and Briones,M. (2010) Leukocyte 6. Carman,C.V. and Springer,T.A. (2003) Integrin avidity adhesion deficiency-III in an African-American regulation: are changes in affinity and conformation patient. Pediatr. Blood Cancer, 55, 180-182. underemphasized? Curr. Opin. Cell Biol., 15, 547-556. 13. Kuijpers,T.W., van Bruggen,R., Kamerbeek,N., 7. Shen,B., Delaney,M.K. and Du,X. (2012) Inside- Tool,A.T., Hicsonmez,G., Gurgey,A., Karow,A., out, outside-in, and inside-outside-in: G protein Verhoeven,A.J., Seeger,K., Sanal,O. et al. (2007)
130 Natural history and early diagnosis of LAD-1/ architecture dictates three-dimensional migration variant syndrome. Blood, 109, 3529-3537. modes of human macrophages: differential 14. Svensson,L., Howarth,K., McDowall,A., Patzak,I., involvement of proteases and podosome-like Evans,R., Ussar,S., Moser,M., Metin,A., Fried,M., structures. J. Immunol., 184, 1049-1061. Tomlinson,I. et al. (2009) Leukocyte adhesion 25. Chiaradonna,F., Fontana,L., Iavarone,C., deficiency-III is caused by mutations in KINDLIN3 Carriero,M.V., Scholz,G., Barone,M.V. and affecting integrin activation. Nat. Med., 15, 306-312. Stoppelli,M.P. (1999) Urokinase receptor-dependent 15. Jurk,K., Schulz,A.S., Kehrel,B.E., Rapple,D., and -independent p56/59(hck) activation state is Schulze,H., Mobest,D., Friedrich,W.W., Omran,H., a molecular switch between myelomonocytic cell Deak,E., Henschler,R. et al. (2010) Novel integrin- motility and adherence. EMBO J., 18, 3013-3023. dependent platelet malfunction in siblings with 26. Schachtner,H., Calaminus,S.D., Thomas,S.G. leukocyte adhesion deficiency-III (LAD-III) and Machesky,L.M. (2013) Podosomes in caused by a point mutation in FERMT3. Thromb. adhesion, migration, mechanosensing and matrix Haemost., 103, 1053-1064. remodeling. Cytoskeleton (Hoboken. ). 16. van de Vijver., Maddalena,A., Sanal,O., Holland,S.M., 27. Dovas,A. and Cox,D. (2011) Signaling networks Uzel,G., Madkaikar,M., de Boer,M., van,L.K., regulating leukocyte podosome dynamics and 6 Koker,M.Y., Parvaneh,N. et al. (2012) Hematologically function. Cell Signal., 23, 1225-1234. important mutations: leukocyte adhesion deficiency 28. Cervero,P., Panzer,L. and Linder,S. (2013) (first update). Blood Cells Mol. Dis., 48, 53-61. Podosome reformation in macrophages: assays 17. Roos,D. and de Boer,M., (1986) Purification and and analysis. Methods Mol. Biol., 1046, 97-121. cryopreservation of phagocytes from human 29. Schmidt,S., Moser,M. and Sperandio,M. (2013) blood. Methods Enzymol., 132, 225-243. The molecular basis of leukocyte recruitment and 18. Cougoule,C., Carreno,S., Castandet,J., Labrousse,A., its deficiencies. Mol. Immunol., 55, 49-58. Astarie-Dequeker,C., Poincloux,R., Le Cabec,V., 30. Robert,P., Canault,M., Farnarier,C., Nurden,A., and Maridonneau-Parini,I. (2005) Activation of the Grosdidier,C., Barlogis,V., Bongrand,P., Pierres,A., lysosome-associated p61Hck isoform triggers the Chambost,H. and Alessi,M.C. (2011) A novel biogenesis of podosomes. Traffic., 6, 682-694. leukocyte adhesion deficiency III variant: kindlin-3 19. Riedl,J., Crevenna,A.H., Kessenbrock,K., Yu,J.H., deficiency results in integrin- and nonintegrin- Neukirchen,D., Bista,M., Bradke,F., Jenne,D., related defects in different steps of leukocyte Holak,T.A., Werb,Z. et al. (2008) Lifeact: a versatile adhesion. J. Immunol., 186, 5273-5283. marker to visualize F-actin. Nat. Methods, 5, 605-607. 31. Becker,H.M., Rullo,J., Chen,M., Ghazarian,M., 20. Cougoule,C., van Goethem,E., Le Cabec,V., Bak,S., Xiao,H., Hay,J.B. and Cybulsky,M.I. (2013) Lafouresse,F., Dupre,L., Mehraj,V., Mege,J.L., alpha1beta1 integrin-mediated adhesion inhibits Lastrucci,C. and Maridonneau-Parini,I. (2012) macrophage exit from a peripheral inflammatory Blood leukocytes and macrophages of various lesion. J. Immunol., 190, 4305-4314. phenotypes have distinct abilities to form 32. Lund,S.A., Wilson,C.L., Raines,E.W., Tang,J., podosomes and to migrate in 3D environments. Giachelli,C.M. and Scatena,M. (2013) Osteopontin Eur. J. Cell Biol., 91, 938-949. mediates macrophage chemotaxis via alpha4 21. Kuijpers,T.W., van Bruggen.R., Kamerbeek,N., and alpha9 integrins and survival via the alpha4 Tool,A.T., Hicsonmez,G., Gurgey,A., Karow,A., integrin. J. Cell Biochem., 114, 1194-1202. Verhoeven,A.J., Seeger,K., Sanal,O. et al. (2007) 33. Ley,K., Laudanna,C., Cybulsky,M.I. and Natural history and early diagnosis of LAD-1/ Nourshargh,S. (2007) Getting to the site of variant syndrome. Blood, 109, 3529-3537. inflammation: the leukocyte adhesion cascade 22. Cohen,S.J., Gurevich,I., Feigelson,S.W., Petrovich,E., updated. Nat. Rev. Immunol., 7, 678-689. Moser,M., Shakhar,G., Fassler,R. and Alon,R. (2013) 34. Alblas,J., Ulfman,L., Hordijk,P. and Koenderman,L. The integrin coactivator Kindlin-3 is not required for (2001) Activation of Rhoa and ROCK are essential lymphocyte diapedesis. Blood, 122, 2609-2617. for detachment of migrating leukocytes. Mol. 23. van de Vijver,E., van den Berg,T.K. and Kuijpers,T.W. Biol. Cell, 12, 2137-2145. (2013) Leukocyte adhesion deficiencies. Hematol. 35. Palecek,S.P., Schmidt,C.E., Lauffenburger,D.A. Oncol. Clin. North Am., 27, 101-16, viii. and Horwitz,A.F. (1996) Integrin dynamics on the 24. van Goethe,E., Poincloux,R., Gauffre,F., tail region of migrating fibroblasts. J. Cell Sci., 109 Maridonneau-Parini,I. and Le Cabec,V., (2010) Matrix ( Pt 5), 941-952.
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Summary and general discussion 7
7.1 cd47-SIRPα interactions control phagocyte effector functions towards host cells By means of their effector functions, phagocytes contribute to the detection and elimination of pathogens, induction and resolution of inflammation and maintenance of homeostasis. To do so, phagocytes sense and integrate information from their environment using, among other mechanisms, transmembrane protein receptors that upon ligation transduce signals into the cell. One of these transmembrane proteins expressed on phagocytes is SIRPα, an inhibitory glycoprotein expressed selectively on immune cells and the nervous system. SIRPα interacts with the broadly expressed counter-receptor CD47 and has been found to regulate several functions relevant in the context of homeostasis and inflammation. These include cell adhesion and migration, were it has been found to regulate leukocyte transmigration (1;2) and the dynamics of the cytoskeleton (3). A link between integrin activation and SIRPα phosphorylation has been 7 established as well, as integrin ligation can trigger phosphorylation of SIRPα and induce it to act as a scaffold protein. In this context it can signal for example through SKAP2 to control actin dynamics in macrophage phagocytosis (3). Another well characterized function of SIRPα is its role in host cell phagocytosis. Initially discovered to control the uptake by macrophages of RBCs and platelets, it has been later revealed that SIRPα-CD47 interactions regulate the uptake of many hematopoietic cell types including hematopoietic stem cells, as well as cancer cells (5,6). Furthermore CD47 is upregulated on circulating mobilized HSC, and plays a role in the clearing of this particular pool of HSC by macrophages in vivo (4). As such SIRPα-CD47 interactions are instrumental for improving human engraftments on immune-compromised mouse models. The SIRPα-CD47 interaction is generally lost across species and reestablishing it either by allele selection (the NOD SIRPα allele provides very good binding to human CD47, thus explaining the superiority of this background in supporting human cell transplantation) or artificial means (overexpression, knock-in) improves many engraftment models by increasing the lifespan of the implant (8,9). This discovery opened a door to improve xenotransplantation in humans were animal tissues could be genetically modified to express CD47 alleles that interact with human SIRPα. In line with this transgenic overexpression of human CD47 in animals, such as pigs that are anatomically suitable for the transplantation of many organs into patients, is a promising strategy to improve the efficacy of xenotransplantation. In the field of adaptive immunity activation SIRPα has been discovered to regulate DC and T cells functions. Regarding DCs from the SIRPα mutant mouse, several studies point to a defect in their ability to trigger Th1, Th2, Th17 and NKT cell responses (10-22). The mechanism(s) behind this are not totally clear, but it might involve modulation of DC migration, maturation or/and antigen presentation. Related to this last point we have to mention that reduced levels of CD4 T cells are found in the SIRPα mice, which could be the result of poor T cell priming by DC(5). SIRPα has been found to play a role in cancer, were high levels of CD47 expressed on cancer cells were shown to be an adverse prognosis factor possibly by inhibiting uptake by macrophages (7). These findings have led to the design of strategies to target the SIRPα-CD47 interaction in
135 the fight against cancer, especially in synergy with antibody therapy. Disturbing this interaction raises of course concerns, being the most obvious the possible induction of autoimmunity by masking CD47 or inhibiting SIRPα signaling. It should be mentioned that neither genetic interference with CD47 or SIRPα in mice nor treatment in different animal species, including primates, with CD47 or SIRPα antagonists showed evidence of overt autoimmunity or any other significant side effects (24,25). Actually it turns out that SIRPα signaling is required for the induction autoimmune responses in certain models of autoimmunity in mice, like experimental autoimmune encephalomyelitis (EAE) (13,26), bacterial- or collagen- induced arthritis (CIA) (6-8) or colitis (9). In these cases the evidence again points to a role for SIRPα in regulating T cell priming by DCs which are essential to develop autoimmune responses in the mentioned models. Few studies have explored the role of SIRPα in infection and inflammation in vivo but a recent study by Li XL et. al has shown susceptibility of the SIRPα mutant mice to infection with an attenuated Salmonella typhimurium strain (10). In this model the phenotypic difference emerged only relatively late after infection and could be attributed to defective Salmonella- specific T and B cell responses, thereby indirectly excluding a substantial role for SIRPα in innate anti-bacterial host defence, at least in this model.
7.2 on the function of SIRPα in the immune system: beyond the expected The above pictures CD47-SIRPα interactions as the core of a homeostatic mechanism by which the ‘self’ molecule CD47, which is broadly expressed on host cells, interacts with SIRPα on phagocytes thereby restricting their effector functions towards the host. The aim of the studies described in this thesis was to explore other potential roles of SIRPα and other SIRP family members in the regulation of phagocyte function. In particular, we have identified novel roles in i) the regulation of phagocyte migration, ii) the control over the phagocyte anti-microbial NADPH oxidase, iii) the interaction between phagocytes and poxviral infected cells, and iv) the triggering of acute myeloid leukemic cell apoptosis that may be exploited as a novel method for therapeutic intervention in leukemia patients. In the following sections I will summarize and discuss in general terms the results obtained in each of the preceding chapters. Furthermore, these findings will be discussed in the context of the recent interest in therapeutic targeting of CD47-SIRPα interactions in cancer.
7.2.1. SIRPα and the integrin activation regulator kindlin3 in phagocyte migration Exploration of the role of SIRPα in adhesion and migration was prompted by the discovery of enhanced actin stress fiber formation in and reduced migration capacity of fibroblasts from the SIRPα mutant mice (11). Furthermore, several studies have provided in vitro evidence for SIRPα regulating the transendothelial- and epithelial- migration of neutrophils and monocytes upon ligation with CD47 (1;2;12). However, both the contributions of CD47 and SIRPα to
136 phagocyte migration in vivo, as well as the exact way(s) in which each of these two proteins regulate the extravasation process had remained unclear. In chapter 5 we report studies describing the role of SIRPα signaling in phagocyte migration in vivo in an acute inflammation model (i.e. thioglycolate-induced peritonitis). Although we did find a significant difference in the kinetics by which both neutrophil and monocyte migration between wild type and SIRPα mutant phagocytes infiltrated the peritoneum, this difference was not very pronounced and the highest numbers of phagocytes in the peritoneum at the peak of the response were similar demonstrating that the overall magnitude of the inflammatory response was not affected. Furthermore, we could not precisely establish which step(s) of the migration process were affected by SIRPα signaling during the in vivo migration. On the other hand we did manage to identify a role for SIRPα signaling specifically with respect to the amoeboid type of migration in vitro. As these experiments were not performed in a tissue context, which at least excludes trans interactions between SIRPα and CD47, they point to a cell-autonomous and potentially CD47-independent role for SIRPα signaling in macrophage migration. The significance of these results in the context of microbial infection would have to be further investigated, but 7 published studies in which SIRPα mutant mice were subjected to Salmonella typhimurium infection showed no differences in susceptibility on the early phase of infection, consistent with a minor role, if any, for SIRPα on the regulation of innate immune responses.As indicated above, the SIRPα mutant mice did show an increased outgrowth of bacteria upon 2-3 weeks after the infection, but these results could primarily be explained by a reduction of CD4 Th1 responses to Salmonella-specific antigens, inefficient antigen presentation, and/or reducedSalmonella - specific Ab formation (see also the discussion in section 7.2.2). It appears feasible that SIRPα plays a role in CD4 T cell priming by DC, for instance as a result of impaired DC migration (13). Whether this also underlies the decreased numbers of CD8α -, CD11c+ DC that have been observed in the peripheral lymphoid tissues of SIRPα mutant mice is not known (14;15). We have also studied the role of the integrin regulator protein kindlin3 in the context of macrophage adhesion and migration. Of interest with respect to SIRPα function our unpublished findings (Matlung and Zhao et al., unpublished) show that neutrophil killer synapse formation during antibody-dependent destruction of tumor cells requires kindlin3-dependent CD11b/ CD18 integrin activation that can be counteracted by CD47-SIRPα interactions and signaling, thus suggesting that SIRPα signaling and kindlin3-mediated integrin regulation somehow converge in phagocytes. Previous studies had already demonstrated that kindlin3 plays an essential role in the activation of integrins in a variety of other hematopoietic cells as well, and also plays an essential role in the extravasation of neutrophils and lymphocytes, thereby explaining the leukocyte adhesion deficiency syndrome in patients lacking kindlin3. To study the contribution of kindlin3 to macrophage trafficking, which had not been explored before and which could potentially be regulated in a (partly) different fashion, we have analyzed the migration properties of monocytes from two unrelated LADIII patients that carry mutations that result in a total absence of the kindlin3 protein. We have found LADIII monocytes to be strongly impaired in their capacity to extravasate from the circulation. Furthermore, macrophages from LADIII patients have substantial defects in adhesion and spreading. On the other hand
137 these cells were able to perform chemotaxis and to migrate in 2D and 3D environments in a relatively normal fashion. In order to adhere, macrophages create a specialized type of adhesion structure termed the “podosome”. In absence of kindlin3 these podosomes lose their characteristic anatomy and also the dynamics of their formation and degradation are altered. While podosomes are thought to be essential for the mesenchymal mode of migration, were they are assumed to orient lysosomal vesicle-mediated release of proteolytic activity required for degrading the interstitial tissue during migration, the LADIII cells migrated normally despite their profoundly disturbed podosomes. The present study provides insight into the formation of these complicated adhesion structures and in particular into the role of kindlin3 in this process. It also raises interesting questions with respect to the role of integrins and their activation during the different modes of migration of macrophages. One possibility is that these cells can migrate normally under conditions of little or no integrin activation, or that they have alternative kindlin3-independent routes for integrin activation. Our recent studies with neutrophils have also shown that some stimuli can induce kindlin3-independent CD11b/CD18- mediated adhesion (16), and collectively these studies show that leukocyte integrin activation may be regulated in a more complex way than previously anticipated.
7.2.2. SIRPα and the NADPH oxidase Although CD47-SIRPα interactions play a pivotal role in restricting immunity towards host cells as outlined above, it was not known whether phagocyte effector functions involved in the defense against microbial infection were perhaps also regulated by SIRPα signaling in phagocytes. One of the major anti-microbial effector mechanisms employed against bacterial and fungal infection(s) is the phagocyte NADPH oxidase. The importance of the NADPH oxidase for the host defense is illustrated by the increased susceptibility against bacterial and fungal infection in chronic granulatomous disease patients that lack the functional enzyme (17;18). In chapter 2 we describe how SIRPα overexpression can regulate the activity of the phagocyte NADPH oxidase by selectively downregulating the expression levels of gp91phox in vitro. This appeared to require so called cis interactions between CD47 and SIRPα on the cell surface of the myeloid cells as well as signaling via the ITIM motifs of the SIRPα cytoplasmic tail. Although consistent with these findings we did find an increased oxidase activity on myeloid cells form the SIRPα mutant mice, we could not find a role for SIRPα in a short term, 5 day, in vivo infection model with Salmonella typhimurium, suggesting that the overall effect of a SIRPα signaling defect has little consequence for the overall innate component of the host defence against Salmonella. The latter model was chosen because the NADPH oxidase is known to be part of the host response against Salmonella at the early stages of the infection in this model (19). Nevertheless, there is evidence for a contribution of SIRPα later during infection, which is most likely mediated by modulation of adaptive immunity as discussed above. It should be mentioned there is actually no other reported evidence for a putative role for SIRPα in regulating innate immune responses in vivo. Therefore the relevance of SIRPα in innate host defense, if any, remains to be established. It is relevant to mention that whereas we found the lack of SIRPα signaling to have no effect on the production of inflammatory mediators by macrophages other studies have claimed that
138 there is a prominent negative role. In particular an enhanced production of NO, IL6 and TNF-α by macrophages in which SIRPα was knocked-down was shown (33). This apparent discrepancy with our own experiments might be explained by the use of siRNA or shRNA-mediated knock- down strategies, which may have triggered danger pathways thereby affecting cellular activation status. We feel that our model, were we made use of genetic manipulation, is a better tool to study such responses so we believe that the contribution of SIRPα in regulating the production of inflammatory mediators by innate immune cells is negligible.
7.2.3. SIRPα and programmed cell death triggering in AML A lot of research has been conducted in the last few years concerning SIRPα-CD47 interactions and their role in the elimination of cancer cells. Basically it has been found that many cancer types and specially cancer stem cells upregulate CD47 and that this avoids clearance by macrophages or other means (7). In most cases the targeting of CD47-SIRPα interactions does not enhance phagocytosis by macrophages by itself but it does improve antibody-based cancer therapy in 7 a variety of models (20). Nevertheless, there are also some indications that CD47 in cancer cells affects the behavior of the cancer cells themselves (21). In Chapter 3 we have explored the possibility of SIRPα, which is often expressed on cancer cells, to be instrumental as a potential therapeutic target on acute myeloid leukemic cells. We show that AML patients with high expression of SIRPα on their leukemic cells showed poor prognosis and that expression levels of SIRPα were determined by the maturation state of the leukemia. Furthermore, we describe that SIRPα levels were directly enhanced upon differentiation of AML cell lines and that ligation of SIRPα by a monoclonal Ab induced a caspase-independent type of programmed cell death (PCD). Finally, the killing of AML cell lines by SIRPα ligation synergized with established anti- leukemic drugs, demonstrating the possible use of this targeted strategy in combination with established agents. While most of the attention in the area of cancer is focused on interfering with the SIRPα-CD47 interactions, we think it might be useful to further explore the current concept of PCD induction through SIRPα in the context of AML in particular.
7.2.4. SIRPs and Immune evasion As a family of paired receptors, the various SIRP family members and in particular SIRPα, SIRPβ1 and SIRPγ show a high level of similarity on their extracellular domains. The different genes within the SIRP family clearly emerged from the founding member, SIRPα, by means of gene duplication events and diversification. Like members of other paired receptor families some of the SIRPs like SIRPα and SIRPβ1 show very high levels of genetic variability both between species as well as within a given species. Normally, like in the case of other paired receptors like KIRs, Ly49 or MHC, these polymorphic proteins bind to polymorphic ligands, but in the case of SIRPα the binding partner is CD47, a very well conserved protein. If is not the polymorphic nature of the ligand, what could be the evolutionary force driving the generation of such diversity? We and others propose that pathogen pressure might be responsible for the variability observed in SIRPα and the generation of other members of its family (22;23). Interestingly, all poxviruses express a CD47 homologue (vCD47), that has apparently been hijacked from the host. Indeed, in
139 vivo studies with the rabbit myxoma poxvirus suggest an important immunosuppressive function for vCD47. In chapter 4 we explore the possibility of the human poxvirus variola vCD47 binding to one or more members of the human SIRP family. Although we did not find final prove of which members of the SIRP family are involved in the interaction with vCD47 we did find binding of this molecule to myeloid cells and this interaction could be selectively inhibited by certain monoclonal Abs raised against SIRP family members. Intruighingly, however, ectopic expression of individual SIRP family members or combinations thereof was not sufficient to confer vCD47 binding, suggesting that another factor e.g. a myeloid-specific posttranslational modification of SIRP family members, or a myeloid-specific co-receptor was required as well. Further studies are required to understand the specificity and biochemical basis for this interaction. Based on these findings we hypothesized that Variola virus-infected cells will express vCD47 to bind SIRPα in myeloid cells and inhibit the phagocytosis and/or killing by phagocytes. Polymorphisms in SIRPα may have occurred and been selected to avoid such interactions, while preserving the homeostatically relevant binding to the endogenous CD47. Following the same principle activating members of the family like SIRPβ1 might have evolved as viral receptors that do not bind endogenous CD47 but that might bind vCD47. Further insight into this might clearly be of special interest in the context of understanding the existing diversity of the SIRP family members (SNPs, family members and copy number variations) within human populations. On the other hand it remains also possible that poxviruses employ their vCD47 to modify other host functions, like regulating integrins or that they have adopted other functions not related to the original role of CD47, as has also been reported for other genes hijacked by viruses (24).
7.3. final remarks Taken together, the studies described in this thesis have identified a number of previously unknown functions of the inhibitory SIRPα immunoreceptor in phagocytes (Figure 1). As such they provide further insight into the pleiotrophic nature of this intriguing receptor in the immune system. Work presented in recent years by our group and those of others have shown that interactions between CD47 and SIRPα are a very promising target for therapeutic intervention in particular for potentiating the clinical efficacy of antibody therapy in cancer. This may be important for reducing the need for non-specific chemotherapeutics for the treatment of metastatic or other disseminated forms of cancer. An important question in this context is clearly whether interference with CD47-SIRPα interactions will cause autoimmunity or otherwise harmful side effects. Together with research performed by many other groups (4;20), which have only identified minor effects of CD47-SIRPα manipulation on circulating red blood cell counts, the results described in this thesis support an important and versatile role of SIRPα in the regulation of phagocyte effector functions, while at the same time they do not raise major concerns for therapeutic targeting of the CD47-SIRPα pathway.
140 Figure 1. SIRPα signal transduction upon CD47 binding. SIRPα–CD47 interactions trigger the recruitment of phosphatases like SHP1 and SHP2 to the ITIMs of SIRPα resulting in their activation and the dephosphorylation of different target substrates which in turn regulate cell functions. The oval with the question mark represents other possible adaptor or signaling molecules that could interact with the cytoplasmic tail of SIRPα and contribute to 7 signal transduction (3) .
References 1. Liu,Y., Buhring,H.J., Zen,K., Burst,S.L., Schnell,F.J., Okazawa,H., Ohnishi,H. et al. (2008) Resistance to Williams,I.R. and Parkos,C.A. (2002) Signal collagen-induced arthritis in SHPS-1 mutant mice. regulatory protein (SIRPalpha), a cellular ligand Biochem. Biophys. Res. Commun., 371, 561-566. for CD47, regulates neutrophil transmigration. J. 7. Tanaka,K., Horikawa,T., Suzuki,S., Kitaura,K., Biol. Chem., 277, 10028-10036. Watanabe,J., Gotoh,A., Shiobara,N., Itoh,T., 2. de Vries,H.E., Hendriks,J.J., Honing,H., De Yamane,S., Suzuki,R. et al. (2008) Inhibition of Src Lavalette,C.R., van der Pol,S.M., Hooijberg,E., homology 2 domain-containing protein tyrosine Dijkstra,C.D. and van den Berg,T.K. (2002) Signal- phosphatase substrate-1 reduces the severity regulatory protein alpha-CD47 interactions are of collagen-induced arthritis. J. Rheumatol., 35, required for the transmigration of monocytes across 2316-2324. cerebral endothelium. J. Immunol., 168, 5832-5839. 8. Verdrengh,M., Lindberg,F.P., Ryden,C. and 3. Alenghat,F.J., Baca,Q.J., Rubin,N.T., Pao,L.I., Tarkowski,A. (1999) Integrin-associated protein Matozaki,T., Lowell,C.A., Golan,D.E., Neel,B.G. (IAP)-deficient mice are less susceptible to and Swanson,K.D. (2012) Macrophages require developing Staphylococcus aureus-induced Skap2 and Sirpalpha for integrin-stimulated arthritis. Microbes. Infect., 1, 745-751. cytoskeletal rearrangement. J. Cell Sci.. 9. Fortin,G., Raymond,M., Van,V.Q., Rubio,M., 4. Jaiswal,S., Jamieson,C.H., Pang,W.W., Park,C.Y., Gautier,P., Sarfati,M. and Franchimont,D. (2009) A Chao,M.P., Majeti,R., Traver,D., Van,R.N. and role for CD47 in the development of experimental Weissman,I.L. (2009) CD47 is upregulated on colitis mediated by SIRPalpha+C. J. Exp. Med., 206, circulating hematopoietic stem cells and leukemia 1995-2011. cells to avoid phagocytosis. Cell, 138, 271-285. 10. Li,L.X., Atif,S.M., Schmiel,S.E., Lee,S.J. and 5. Saito,Y., Iwamura,H., Kaneko,T., Ohnishi,H., McSorley,S.J. (2012) Increased susceptibility to Murata,Y., Okazawa,H., Kanazawa,Y., Sato- Salmonella infection in signal regulatory protein Hashimoto,M., Kobayashi,H., Oldenborg,P.A. et alpha-deficient mice. J. Immunol., 189, 2537-2544. al. (2010) Regulation by SIRPalpha of dendritic cell 11. Inagaki,K., Yamao,T., Noguchi,T., Matozaki,T., homeostasis in lymphoid tissues. Blood, 116, 3517- Fukunaga,K., Takada,T., Hosooka,T., Akira,S. and 3525. Kasuga,M. (2000) SHPS-1 regulates integrin- 6. Okuzawa,C., Kaneko,Y., Murata,Y., Miyake,A., mediated cytoskeletal reorganization and cell Saito,Y., Okajo,J., Tomizawa,T., Kaneko,Y., motility. EMBO J., 19, 6721-6731.
141 12. Liu,Y., O’Connor,M.B., Mandell,K.J., Zen,K., Ullrich,A., current management of chronic granulomatous Buhring,H.J. and Parkos,C.A. (2004) Peptide- disease. Hematol. Oncol. Clin. North Am., 2, 253-266. mediated inhibition of neutrophil transmigration by 18. Roos,D. (1994) The genetic basis of chronic blocking CD47 interactions with signal regulatory granulomatous disease. Immunol. Rev., 138, 121-157. protein alpha. J. Immunol., 172, 2578-2585. 19. van Diepen,A., van der Straaten,T., Holland,S.M., 13. Baba,N., Van,V.Q., Wakahara,K., Rubio,M., Fortin,G., Janssen,R. and van Dissel,J.T. (2002) A superoxide- Panzini,B., Soucy,G., Wassef,R., Richard,C., Tamaz,R. hypersusceptible Salmonella enterica serovar et al. (2013) CD47 fusion protein targets CD172a+ cells Typhimurium mutant is attenuated but regains in Crohn’s disease and dampens the production of IL- virulence in p47(phox-/-) mice. Infect. Immun., 1beta and TNF. J. Exp. Med., 210, 1251-1263. 70, 2614-2621. 14. Sato-Hashimoto,M., Saito,Y., Ohnishi,H., 20. Weiskopf,K., Ring,A.M., Ho,C.C., Volkmer,J.P., Iwamura,H., Kanazawa,Y., Kaneko,T., Kusakari,S., Levin,A.M., Volkmer,A.K., Ozkan,E., Fernhoff,N.B., Kotani,T., Mori,M., Murata,Y. et al. (2011) van de Rijn,M., Weissman,I.L. et al. (2013) Engineered Signal regulatory protein alpha regulates the SIRPalpha variants as immunotherapeutic adjuvants homeostasis of T lymphocytes in the spleen. J. to anticancer antibodies. Science, 341, 88-91. Immunol., 187, 291-297. 21. Zhao,X., Matlung,H., Kuijpers,T.W. and van den 15. Okajo,J., Kaneko,Y., Murata,Y., Tomizawa,T., Berg,T.K. (2013) On the mechanism and benefit of Okuzawa,C., Saito,Y., Kaneko,Y., Ishikawa- siRNA-mediated targeting of CD47 in cancer. Mol. Sekigami,T., Okazawa,H., Ohnishi,H. et al. (2007) Ther., 21, 1811. Regulation by Src homology 2 domain-containing 22. Akkaya,M. and Barclay,A.N. (2012) How do protein tyrosine phosphatase substrate-1 of alpha- pathogens drive the evolution of paired galactosylceramide-induced antimetastatic activity receptors? Eur. J. Immunol.. and Th1 and Th2 responses of NKT cells. J. Immunol., 23. Barclay,A.N. and van den Berg,T.K. (2013) The 178, 6164-6172. Interaction Between Signal Regulatory Protein 16. van de Vijver,E., V, Tool,A.T., Sanal,O., Cetin,M., Alpha (SIRPalpha) and CD47: Structure, Function, Unal,S., Aytac,S., Seeger,K., Pagliara,D., Rutella,S., and Therapeutic Target. Annu. Rev. Immunol.. van den Berg,T.K. et al. (2013) Kindlin-3-independent 24. Engel,P., Perez-Carmona,N., Alba,M.M., adhesion of neutrophils from patients with leukocyte Robertson,K., Ghazal,P. and Angulo,A. (2011) adhesion deficiency type III. J. Allergy Clin. Immunol.. Human cytomegalovirus UL7, a homologue of the 17. Forrest,C.B., Forehand,J.R., Axtell,R.A., Roberts,R.L. SLAM-family receptor CD229, impairs cytokine and Johnston,R.B., Jr. (1988) Clinical features and production. Immunol. Cell Biol., 89, 753-766.
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Acknowledgements 8
And so it is, I finished my thesis. It has been a long way on which I have learned even more that what I expected when I started on that 1st of September 2008. During my PhD there have been moments when it felt like I was by my self against the elements, but there are many people who I would like to thank because they have been essential, of much help or siblings on this scientific journey.
First of all I would like to deeply thank Timo and Taco. I still remember my interview for my position at Sanquin. I recall leaving the old building feeling that I had it. Whatever was going to happen on the next 4 years I would manage with supervisors like you. And so it was. Timo, from the first months I have always been thankful of how you supervised my project, I think this is very difficult to do but you knew when to give me space and when to push me. You both have helped me so much with the last steps of my thesis when everything seamed to be slowing down. I want to thank you both for showing me what science is about, it has been a pleasure learning immunology with you both.
Dirk, I am thankful for your comments to my work in the working discussions and for showing me how one must be rigorous and responsible when working on science. You have also more or less founded the lab were I have learned so much and you are going to be the chairman on my defense. I couldn’t think of anyone that would be better for this task. Thank you very much for everything. 8 Karin, you have had to adjust to me more than anyone, and I want to thank you for that. You as a morning person and me as a……well, we all know, found a way to work together and to become friends. Thanks a lot for working hard with me.
Paul, man we had fun in the lab! Countless jokes and joyful spirit in its pure state! But on top of that I thank you for being so good at your job, I loved working with you because you always manage to do the job perfectly and fast. Most importantly I want to thank you for your tenacity during these 5 years on finding my stolen banana, we didn’t succeed, but it doesn’t matter, I know you will keep on trying
Judy and Martin, my DNA gurus! Thanks a lot for been patient with me, you were always there with a smile and a positive attitude to check with me my gel or discuss molecular genetics, I have learned a lot from you and had very good times with both of you.
Laura and Robin, the ambitious upcoming PI’s. I have shared so much fun and cotilleos with you both and you have helped me with practical matters several times. On top of that you both have been very important for me to understand academia and how it works. From both of you I have learned (on two very different styles) what it takes to climb the ambitious ladder of science.
Michel, the last of the SIRP technicians! You have helped me so much too. Always open to help PhD a student in trouble! We have enjoyed those videos we made eh my friend?
Edith, one of my science sisters, but also something more. We both made each others life in the lab nicer, and how much we talked about work! I want to thank you because although you might think it helped you, it also helped me a lot.
Sietse, Roel and Joris, the dudes. It has been so nice to share the PhD with you guys, all of us so different and making such a good connection at the same time. Thanks a lot Joris for picking me as
147 your paranimf, it has helped me push the last bits of my thesis forward! Roel and Sietse, its you turn now to get things done, but since you are lab beasts I am sure you wont have any problem with this.
Xiwen, Katka, Elena and Boukje, the girls. Ah! So nice it was to enter the room everyday and see you all there. Dr. Xiwen, like you I wont forget my first months in the Netherlads when I had a Chinese wife either. We have shared a lot and for that I am thankful! Katka, damn we talked eh? We always complained about Hanke, but I think it was us that talked the most. Thanks for listening! Boukje and Elena, you joined the SIRP room (because it was and it will be a SIRP room) and both your laughs took over, so nice to hear both of them, thanks for all the great moments and talks we had.
Hanke, what can I say. I owe you soooo much. My project had a major turn when you came to Sanquin. I learned so much from your experience, from your engineer cubic mind, from your “lets tackle the problem step by step” and from your animal experience. How many things I have asked you! Millions! You have been like my godmother (I know you won’t like the term but its true) in science, and a very good friend also, that is why I had no doubt when picking you as my paranimf. Thank you so much for everything!
Jean, my man. You have no clue about immunology and still you have been a big part in this PhD. We lived together for so many years during which you always helped me to see problems in perspective when I got negative and to see things from a fresh point of view. Rebooting my feelings towards my PhD project has been essential to finish it and together with my boss you have been very helpful on doing this. On top of that we have rocked Amsterdam hard and as a powerful duo, now you are gone to cheese land but still you will be my paranimf and for that I thank you again.
People of the Klimmuur, Hilda, Erik Irene, Jeanet, Anne and others. I have bothered you so much with my PhD that you definitely deserve a space in this book. All those evenings when we would meet at the klimmuur and I would share my stress with you and burn it by going up and down that wall like maniacs.
Papa, mama y Cata, desde la distancia y quizás por eso mas importante aun, quiero agradeceros la ayuda. Siempre me habéis apoyado en mis decisiones, aunque hayan resultado en que este tan lejos de vosotros en momentos tan dífciles como los que nos ha tocado vivir en los últimos años. Siempre os habeis alegrado de que me haya librado del fiasco en nuestro pais y eso es una expression de amor muy fuerte que me ha ayudado mucho a seguir aqui. Espero que esteis orgullosos de mi por este libro, yo lo estoy!
Cece! I have met you on the last steps of my PhD, still everything I do now I share it with you, including becoming a doctor. The joy I fell when finishing this book is bigger because you feel it too. Soon it will be your turn!
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