Macrophages in the embryo and beyond: Much ... - Wiley Online Library

' 2008 Wiley-Liss, Inc.

genesis 46:447–462 (2008)

REVIEW

Macrophages in the Embryo and Beyond: Much More Than Just Giant Phagocytes Dmitry A. Ovchinnikov* Institute for Molecular Bioscience and Cooperative Research Centre for Chronic Inflammatory Diseases (CRC-CID), University of Queensland, Brisbane, Queensland, Australia Received 21 December 2007; Revised 21 May 2008; Accepted 23 June 2008

Summary: Originally recognized as an essential part of the innate and acquired immune systems, macrophages emerged as omnipresent and influential regulators of embryo- and organo-genesis, as well as of tissue and tumor growth. Macrophages are present essentially in all tissues, beginning with embryonic development and, in addition to their role in host defense and in the clearance of apoptotic cells, are being increasingly recognized for their trophic function and role in regeneration. Some tissue macrophages are also found to posses a substantial potential for autonomous self-renewal. Macrophages are associated with a significant proportion of malignant tumors and are widely recognized for their angiogenesis-promoting and trophic roles, making them one of the new promising targets for cancer therapies. Recent expression profiling of embryonic macrophages from different tissues revealed remarkable consistency of their gene expression profiles, independent of their tissue of origin, as well as their similarities with tumorassociated macrophages. Macrophages are also capable of fusion with other cells in tissue repair and metastasizing tumors, as well as with each other in the immune response and osteoclastogenesis. genesis C 2008 Wiley-Liss, Inc. 46:447–462, 2008. V Key words: embryonic macrophages; definitive macrophages; tumor-associated macrophages; apoptosis; cellcell fusion; fluorescent reporters; Gal4-UAS system

INTRODUCTION In addition to providing general background information on the macrophage lineage and its origins, this review will highlight recent advancements in our understanding of the role of this prominent cell type in many important biological processes. Macrophages are being increasingly recognized for their role in providing trophic support in many tissues, their ability to serve as partners in homoand hetero-typic cell fusions, and for their pivotal role in tumor angiogenesis and metastasis. Macrophages and their precursors were recently found to have a substantial competence in the promotion of, and contribution to, tissue regeneration and repair. This review will also discuss a number of recently generated transgenic tools,

which allow the visualization of macrophages and related cells, their ablation, and manipulation of gene function (by conditional gene inactivation or expression) in the macrophage lineage. The term ‘‘macrophage’’ was introduced in the late nineteenth century by Ilya Mechnikov (Metchnikoff, 1893), and translates from the Greek literally as ‘‘large eaters’’. The very first observations of the process of phagocytosis in an embryonic setting were also made by Mechnikov, in the early 1880’s, when he described an uptake of foreign objects in starfish larvae by amoeba-like cells, which he called phagocytes, ‘‘devouring cells’’ (Tauber, 1991). In this review, the term macrophage will be used, somewhat loosely, to describe both primitive early embryonic phagocytes and definitive macrophages found in the embryos and adult organisms, at later stages. Specialized phagocytes are found in embryos of organisms ranging from Drosophila to higher vertebrates, while in lower organisms this function is relegated to facultatively phagocytic cells, mostly of mesenchymal origin. Even today, the exact origin of some embryonic phagocytes (a term often used interchangeably with ‘‘embryonic macrophages’’) is unclear, therefore it has to be noted that their classification is largely based on the ability to participate in phagocytosis, and on the expression of enzymatic or surface markers. The identity of macrophages is also inferred from the expression of reporter transgenes and transcripts from promoters of genes thought to be macrophage-specific (visualized via in situ hybridization), and on the morphology and behavior of these cells (Lichanska and Hume, 2000; Morris Additional Supporting Information may be found in the online version of this article. * Correspondence to: Dmitry A. Ovchinnikov, Institute for Molecular Bioscience and Cooperative Research Centre for Chronic Inflammatory Diseases (CRC-CID), University of Queensland, Brisbane 4072, Australia. E-mail: [email protected] Contract grant sponsors: Australian Cooperative Research Centre for Chronic Inflammatory Diseases (CRC-CID), University of Queensland Research Development Grant Scheme. Published online 9 September 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/dvg.20417

448

OVCHINNIKOV

et al., 1991; Ovchinnikov et al., 2008; Sasmono et al., 2003). A recent study utilizing cell type-specific ablation in Xenopus laevis highlighted the importance of embryonic macrophages for normal development (Smith et al., 2007). Overexpression of a toxic influenza virus-derived ion channel using macrophage-specific Lurp1 promoter (Smith et al., 2002) lead to an efficient ablation of embryonic macrophages in a fraction of transgenic animals, which then displayed significant developmental limb abnormalities and died at metamorphosis. Historically, two different approaches were taken to the classification of embryonic and adult phagocytes (mostly in vertebrates): one based on functional similarity, and another on the ontogeny of these cells. The first approach led to the formulation of the concept of the reticuloendothelial system (Aschoff, 1924), which incorporated phagocytes and other cells of different origins directly involved in the clearance from the bloodstream of both endogenous (e.g., apoptotic cells) and exogenous ‘‘contaminants’’. Since the 1970’s, the preference is largely given to an ontogeny-based classification of macrophages and related cells into the mononuclear phagocyte system (Hume, 2006; Hume et al., 2002; van Furth and Cohn, 1968). The mononuclear phagocyte system encompasses cells of hematopoietic origin, from early committed progenitors via circulating monocytes to tissue macrophages, including specialized types (e.g., Kupffer cells) and antigen-presenting dendritic cells (e.g., Langerhans cells of the skin) (Hume, 2006). Recent findings, however, suggest that the earliest embryonic phagocyte populations might originate before the onset of blood circulation and erythropoiesis, and bypass the stage of the circulating monocyte, in zebrafish (Herbomel et al., 1999, 2001) and embryos of amniotes (Faust et al., 1997; Hume et al., 1995; Lichanska and Hume, 2000). In zebrafish, the first macrophages originate from the mesodermal field giving rise to both endothelial cells and phagocytes, indicating that the reticuloendothelial system concept, particularly in embryos, has its merits.

than the blood island, traditionally recognized as the main site of hematopoiesis (Herbomel et al., 1999). These precursors express L-plastin (leukocyte-specific plastin) and migrate into the yolk sac, where they differentiate. Some of them join the blood circulation, whereas others invade the head mesenchyme in a process dependent on CSF-1, the pivotal macrophage differentiation and survival factor, as this process is disrupted in the panther mutant fish, which lacks the functional CSF-1 receptor (Herbomel et al., 2001). Interestingly, specialized phagocytes in the Drosophila embryo, plasmatocytes, also originate from the anterior head mesoderm (Tepass et al., 1994; Wood and Jacinto, 2007). Plasmatocytes are a cell group orthologous to embryonic phagocytes of vertebrates, and constitute a majority (up to 95%) of all blood cells, hemocytes (Holz et al., 2003; Wood and Jacinto, 2007). Unlike in vertebrates, in Drosophila, embryonic plasmatocytes remain the only phagocytes during the larval stages until metamorphosis (in pathogen-free conditions). At metamorphosis lymph glands release a new wave of hemocytes, which perform remodeling or eliminate those larval structures destined for alteration, including remnants of the lymph glands themselves, which also happen to be the last hematopoietic organs in the Drosophila lifecycle (Holz et al., 2003; Lanot et al., 2001; Wood and Jacinto, 2007). In the mouse embryo, the second population of macrophages represents a mixture of maternal macrophages and the first really embryonic macrophages. The latter cells seem to originate from precursors in the yolk sac mesoderm, which are detected as early as 7.5 dpc (Palis et al., 2001). They are derived from monopotent precursors and bypass the monocyte-like stage, and are observed in the yolk sac, and later on in the embryo proper from 8 dpc (Bertrand et al., 2005). These yolk sac-derived ‘‘primitive’’ embryonic macrophages then go on to invade the anterior embryonic structures by 9 dpc in the mouse (Ovchinnikov et al., 2008), and by 24 hpf in the zebrafish (Herbomel et al., 2001).

EARLY EMBRYONIC PHAGOCYTES

TRANSITION TO DEFINITIVE HEMATOPOIESIS AND MACROPHAGE POPULATIONS

The first population of macrophages is observed in the mouse embryo at the late head fold stage (7.5 dpc) and, somewhat surprisingly, consists entirely of maternally derived, mature macrophages (Bertrand et al., 2005). This fairly scarce population is found in yolk sac mesoderm and adjacent structures, and is thought to play a scavenger role in the absence of the embryo’s own phagocytes. These macrophages express a number of definitive macrophage (i.e., originating from hematopoietic organs and differentiating from monocytic precursor) markers, such as F4/80, and should not be strictly classified as ‘‘embryonic phagocytes’’ (Hume and Gordon, 1983; Shepard and Zon, 2000). In zebrafish embryos, the first macrophage precursors originate in the ventro-lateral mesoderm anterior to the cardiac field, thus located significantly more rostrally

‘‘Definitive’’ embryonic macrophages formed as a part of the definitive hematopoiesis process from myeloid precursors and through the monocyte stage, share more properties in common with their counterparts found in the adult animal than the ‘‘primitive’’ embryonic phagocytes discussed above. Unlike the latter, they express high levels of macrophage markers PU.1, F4/80, S100A8 (MRP8) and S100A9 (MRP-14), just like macrophages of the adult (Lichanska et al., 1999; Lichanska and Hume, 2000). In mice, it was traditionally thought that hematopoietic precursors populate the main site of definitive hematopoiesis in the embryo, the fetal liver, in two waves (Bonifer et al., 1998; Dzierzak et al., 1998). The first wave of precursors originates from the yolk sac at around 8.5–9 dpc, and the second, half a day later, from

MACROPHAGES IN THE EMBRYO AND BEYOND

the aorta-gonads-mesonephros (AGM) region of the embryo, fully dominating in contribution to the progenitor cell population over the first wave after 10 dpc (Dzierzak et al., 1998; Lichanska and Hume, 2000). A significant body of evidence now supports a model in which long-term hematopoietic progenitors, establishing definitive hematopoiesis in the fetal liver, originate intraembryonically, in the para-aortic splanchnopleura (PAS, composed of both mesodermal and endodermal derivatives) and AGM region of the embryo at 9 dpc (Cumano et al., 2001; Cumano and Godin, 2007; Medvinsky and Dzierzak, 1996). It appears likely that early hematopoietic progenitors can than migrate out of the embryo into the yolk sac, and contribute to the significant population of multipotent hematopoietic progenitors residing in that extraembryonic structure up to 10.5–11 dpc, when the fetal liver takes over as a major site of hematopoiesis in the conceptus (Cumano and Godin, 2007; Dzierzak et al., 1998). While harboring a significant population of pluripotent precursors, PAS/AGM regions do not appear to serve as sites of differentiation of hematopoietic lineages, unlike the yolk sac and the fetal liver. Most findings supporting the latest model are based on assessments of the differentiation potential of hematopoietic progenitors isolated from various embryonic structures in various in vitro differentiation and tissue transplantation assays. For instance, it was established that the PAS/AGM region harbors significant number of multipotent progenitors capable of giving rise to cells of the lymphoid lineage, while progenitors from the yolk sac around 8 dpc are limited to the production of myeloid and erythroid lineages (Cumano et al., 1996, 2001). Yolk sacderived precursors were also found to have a limited and transient myelogenic potential in bone marrow grafts reconstitution assays, in contrast to longer-lasting PAS/ AGM-derived precursors (Cumano et al., 2001). Identical results were obtained in classical chick-quail chimera experiments (Dieterlen-Lievre, 1975), when yolk sac and embryonic compartments were separated prior to the establishment of circulation, therefore eliminating the possibility of progenitors being spread by the circulation. It has to be emphasized that the delineation of the progenitors’ tissue of origin in the mouse embryo without using sophisticated lineage tracing or localized cell labeling techniques is somewhat problematic after establishment of the circulation at 8.5 dpc. Significantly, most of the above-mentioned findings on the mechanisms of establishment of definitive hematopoiesis appear to translate well into the human system. Just like in the mouse, at the same time as the first pluripotent progenitors capable of establishing the whole range of hematopoietic lineages were first found in the human embryo, the differentiation potential of the precursors derived from the yolk sac was restricted to the myeloid lineage (Tavian et al., 1999, 2001). It appears plausible that human embryos also possess two types of embryonic phagocytes similar to those in the mouse: yolk sac-derived primitive and definitive embryonic macrophages.

449

VISUALIZING MACROPHAGES IN THE EMBRYO Detection of one or a set of the macrophage-specific markers remains the most reliable characteristic allowing classification of cell(s) as an embryonic macrophage. This is often complemented by observations of the stellar or amoeboid morphology, highly characteristic vacuolarization, and phagocytosing or patrolling behavior (Bertrand et al., 2005; Herbomel et al., 2001; Hume, 2006; Lichanska and Hume, 2000). Among the best established markers for early, ‘‘primitive’’ embryonic phagocytes, predominantly found in the yolk sac of the mouse embryo at 8–11 dpc, are the mRNA or protein product of the CSF-1(M-CSF) receptor gene Csf1r(c-fms), macrophage mannose receptor (CD206) mRNA, myeloid-specific integrin protein CD11b (Mac-1), and microphthalmia transcription factor (mitf) gene product (Lichanska et al., 1999; Lichanska and Hume, 2000). Another recently ‘‘re-discovered’’ in zebrafish marker for early phagocytes is L-plastin, an evolutionarily conserved leukocyte-specific form of a protein involved in the regulation of cytoskeletal dynamics (Herbomel et al., 2001; Lin et al., 1988; Shinomiya et al., 1994). In addition to the above-mentioned markers, ‘‘definitive’’ embryonic macrophages derived from intraembryonic precursors acquire expression of the markers typical for definitive macrophages, including those found in the adult animals, such as surface antigen F4/80 (Emr1), lysozyme M, and PU.1 (Lichanska et al., 1999; Lichanska and Hume, 2000). They also start expressing an array of genes associated with their phagocytic function, such as scavenger receptors: Type I and II in mouse, croquemort (homologue of mammalian CD36) and dSR-CI in Drosophila (Franc et al., 1996; Kodama et al., 1996; Meister and Lagueux, 2003; Ramet et al., 2001), and members of the ATP-binding cassette transporters family, such as ABC1 (Luciani and Chimini, 1996). Two chemotactic factors, S100A8 (MRP-8) and S100A9 (MRP-14), were found to clearly differentiate definitive embryonic macrophages from those originating in the yolk sac, where these genes appear not to be expressed at all (Goebeler et al., 1993; Lichanska et al., 1999; Passey et al., 1999). Another useful approach to labeling of the embryonic macrophage lineage is to utilize lineage-specific promoters (e.g., for a number of marker genes above) to drive a convenient reporter protein, an exogenous enzymatic activity (lacZ) or a peri-vital fluorescent protein (EGFP, DsRed) (summarized in Table 1). Great advantages of this approach include the experimental ease of visualization and an ability to visualize macrophages in live cells and organisms (Fig. 1). The first feature of transgenic lineage labels makes them more amenable to use as a part of high-throughput experiments, such as mutagenesis or compound screens. The ability to observe fluorescent cells in real time allows one to record the behavior of this rather motile cell group in various settings, including in the whole live embryo (especially in Drosophila and aquatic species). Table 1 contains a list

450

EGFP

ECFP

Csf1r(c-fms) (c-fms-EGFP)

Modified Csf1r(c-fms) (Csf1rDPtroGal4/UAS-ECFP)

Nuclear-localized EGFP, expressed from the UAS-NLS-EGFP, a Gal4 responder line. While embryonic expression of this reporter has not been reported, known expression pattern of the PU.1 gene suggests that the reporter allele is likely to be functional during embryogenesis in definitive embryonic macrophages. b

a

(Ovchinnikov et al., 2008)

(Back et al., 2004) Bertrand et al., 2005; Jung et al., 2000 (Sasmono et al., 2003) EGFP EGFP PU.1 (PU.1 EGFP/1) CX3CR1 (CX3CR1 EGFP/1)

Clawed frog Xenopus laevis Mouse Mus musculus

Embryonic, including early, plasmatocytes Embryonic phagocytes, myeloid lineage (incl. granulocytes) Embryonic phagocytes, myeloid lineage (incl. granulocytes) Embryonic phagocytes, some myeloid cells in adult nEGFP EGFP EGFP, DsRed2 EGFP, DsRed1

Fruitfly Drosophila melanogaster

Zebrafish Danio rerio

Embryonic/larval hemocytes Early plasmatocytes, some glial cells nEGFP lacZ, nEGFP

Hemolectin (hml-Gal4) Glial cell missing(gcm) (gcm-lacZ, gcm-Gal4) Hemese (hemese-Gal4) pu.1(spi1) (spi1-EGFP) Lysozyme C (lysC-EGFP,DsRed2) Lurp1 (Lurp1-EGFP,DsRed1)

Embryonic expression not describedb, myeloid, B lymphocytes In embryonic macrophages from 9.5 dpc, subset of monocytes macrophages in adult animals Trophoblast cells, embryonic phagocytes from 9.5 dpc, myeloid, B cells in adult Embryonic phagocytes (precursors) from 8.5 dpc, subset of macrophages in adult

Asha et al., 2003; Bellen et al., 1989 (Goto et al., 2003) Bernardoni et al., 1997; Paladi and Tepass, 2004 (Zettervall et al., 2004) (Ward et al., 2003) (Hall et al., 2007) Smith et al., 2002, 2007 Phagocytosing plasmatocytes embryonic/larval hemocytes, lacZ, nEGFP Collagen IV (Cg-lacZ, Cg-Gal4)

Expression specificity

a

Marker Promoter/gene

Table 1 Transgenic Markers of Embryonic Phagocytes

References

OVCHINNIKOV

of selected transgenic lines in various model organisms shown to be expressed in embryonic phagocytes; for Drosophila, only a few of the better characterized promoter/enhancer traps or engineered transgenes are listed. All of them were generated in, or converted into (see references therein), a configuration expressing a fluorescent protein, making them suitable for the applications listed above.

MACROPHAGES AND PHAGOCYTOSIS Precisely orchestrated patterns of programmed cell death (PCD) are an essential prerequisite of normal development of essentially all metazoans (Penaloza et al., 2006). In vertebrates, some of the most striking examples include the observation that during neural development, between 20 and 80% of originally produced neurons are eliminated (Gordon, 1995), and over 95% of human B cells are eliminated due to gene recombination faults and anti-self receptor expression (Osborne, 1998). Phagocytosis of effete cells by macrophages is typically thought to consist of four major steps: (1) recognition of a cell as being destined for phagocytosis; (2) tethering of the phagocyte to that cell; (3) phagocytosis/ engulfment of the cell; (4) release of pro- or anti-inflammatory signals by the phagocyte. It is thought that Step 4 has limited relevance to the situation of a developing embryo, but it’s important to note that the ability of macrophages to not induce an inflammatory response in this situation is rather important for normal development and tissue homeostasis. It is generally thought that apoptosis and necrosis differ in that the former, PCD, might elicit anti-inflammatory macrophage behavior, while the latter leads to inflammatory macrophage behavior (Huynh et al., 2002; Savill et al., 2002). Significant progress has been made in recent years towards understanding the molecular basis of the recognition of markers of cells undergoing PCD, essential for the first step of their phagocytosis, the process recently dubbed as ‘‘efferocytosis,’’ from the Latin ‘‘effero’’—‘‘to carry out, to bury’’ (Henson and Hume, 2006). A number of molecular determinants present on apoptotic cells, or ‘‘eat me’’ signals, have been identified. Probably the best established of them is phospholipid phosphatidylserine (PS), normally confined to the cytosol-facing, inner lipid layer of the plasma membrane, and translocated into the outer leaflet in cells undergoing PCD (Fadok et al., 1992). The process of PS translocation appears to be facilitated by a known macrophage-specific gene ABC1 (ATP-binding-cassette transporter 1) (Hamon et al., 2000). ABC1 is a mammalian orthologue of the Caenorhabditis elegans gene ced-7, identified in a classic screen for the genes required for genetically programmed cell elimination in the worm embryo (Ellis et al., 1991). Recognition of the PS by phagocytes is thought to be performed by a number of bridging molecules and receptors, with identity of the later remaining a somewhat contentious issue. Inactivation of the mouse

MACROPHAGES IN THE EMBRYO AND BEYOND

451

FIG. 1. Embryonic macrophages visualized using the MacBlue (Csf1rDPtro-Gal4/UAS-ECFP) transgene. (a). At 11 dpc, embryonic macrophages already appear dispersed throughout the embryo, more dense in the anterior portion of the embryo. The highest levels of ECFP fluorescence are obvious in the fetal liver, which, after being populated by hematopoietic precursors, is giving rise to definitive embryonic macrophages. (b). At 12.5 dpc, the number of macrophages in embryonic tissues appears significantly higher, probably due to the appearance of definitive macrophages. (c). Hindlimb of an 11.75 dpc MacBlue embryo (anterior is up). Areas of increased cell death, such as anterior and interdigital necrotic zones, show higher macrophage concentrations, while cartilaginous condensations are devoid of ECFP1 cells. (d). Hindlimb of a MacBlue embryo at 13.5 dpc. Note high macrophage concentration in receding interdigital webbing. (e). Macrophages in the yolk sac of a 14.5 dpc MacBlue embryo appear to be distributed evenly, and many are juxtaposed to the blood vessels. (f). Confocal imaging-based 3D reconstruction of ECFP1 Langerhans (dendritic) cells of the skin. Cell processes project away from the viewer and terminate on the epithelial surface.

gene called PS receptor (psr) lead to a phenotype consistent with the requirement of the gene for phagocytosis of apoptotic cells by macrophages. Specifically, an excess of apoptotic cell bodies was observed in interdigital spaces of mutant limbs, extraneous tissue was present in mutant lungs, which were virtually solid and devoid of alveolae, and excessive tissue was present in the brain and in the developing eye (Li et al., 2003). Although another independently developed psr null mutant also had some apoptotic cell clearance defects (Kunisaki et al., 2004), the third independent knockout did not (Bose et al., 2004). In combination with reports that the protein actually appears to be localized to the nucleus, and the binding of a monoclonal antibody

found to bind macrophages in PS-inhibitable fashion did not appear to be eliminated in the third knockout, raising doubts about the true function of the psr gene product (Williamson and Schlegel, 2004). Recently, two other putative PS receptors were identified as two members of the Tim (T-cell immunoglobulin- and mucin-domain-containing molecule) family of type I transmembrane proteins, Tim1 and Tim4 (Miyanishi et al., 2007). Tim4 was identified as an antigen for an antibody blocking the PSdependent phagocytosis of apoptotic cells, and both Tim1 and Tim4 were found to specifically bind PS and enhance the phagocytosing ability of the cells overexpressing either of the proteins (Miyanishi et al., 2007). Among the bridge molecules, secreted by macrophages

452

OVCHINNIKOV

and thought to interact with both PS and signaling receptors on the phagocyte membrane, are products of the milk fat globule epidermal growth factor VIII (MFGE8), its homologue developmental endothelial locus-1 (Del-1), and growth arrest-specific 6 (Gas6) genes (Hanayama et al., 2004b; Nakano et al., 1997). MFG-E8 and Del-1 are both thought to interact with the avb3 integrin on the phagocyte surface, and appear to be differentially expressed by various macrophage populations (Hanayama et al., 2002, 2004a). Gas6 is a known ligand for the receptor tyrosine kinases Axl, Mertk, and Tyro3, which have been shown to play important roles in apoptotic cell clearance, with Mertk important for phagocytosis by macrophages, and the other two mostly utilized by the dendritic cells, which are also considered to be a part of the mononuclear phagocyte system (Scott et al., 2001; Seitz et al., 2007). Annexins, another well known family of proteins binding to PS with high affinity (annexin A5, often called annexin V, in particular) is not thought to act as a receptor but rather compete with PS recognition machinery on the phagocyte (Bondanza et al., 2004). An anti-coagulative shield formed by annexin A5 on the surface of PS-rich dead cells (Appelt et al., 2005) is thought to be important in the maintenance of a balance between efferocytosis by macrophages and the immune response-inducing uptake and processing by the dendritic cells, often leading to an autoimmune response (Gaipl et al., 2007). Another likely marker of an apoptotic cell is calreticulin, normally an ER-associated protein overrepresented on the membrane of cells undergoing PCD and recognized by the low density lipoprotein receptor-related protein (LRP-1, CD91) on the phagocyte’s surface (Gardai et al., 2005), or by thrombospondin acting as a bridge between calreticulin and CD36 or av-integrins, e.g., avb3 (Goicoechea et al., 2000). DNA exposed as a result of nuclear fragmentation is also likely to serve as one of the ‘‘eat me’’ signals (Palaniyar et al., 2004). To ensure that normal, viable cells are not attacked by phagocytes, which can constitute up to 15% of the total cell number in some tissues (Henson and Hume, 2006), one would expect them to display a number of inhibitory, ‘‘don’t eat me’’ signals. One of those signals is transmitted by a homotypic interaction between CD31 (PECAM-1) molecules on viable cells and the phagocytes (Brown et al., 2002). Another such signal is likely to be the ‘‘self’’-antigen, CD47, also known as the integrinassociated protein (Gardai et al., 2006; Oldenborg et al., 2000). It is postulated that CD47 present on normal cells interacts with and stimulates the phagocytosis-inhibiting receptor, SIRPa, known to be highly expressed on macrophages, thus antagonizing PS recognition and the subsequent activation of the Src kinase pathway, thought to be responsible for the engulfment-related cytoskeletal rearrangements (Gardai et al., 2006). A number of recent studies identified yet another PS receptor, brain-specific angiogenesis inhibitor-1, a seven transmembrane domain receptor belonging to the family of adhesion-type G-protein-coupled receptors, and the

molecules transmitting its signal inside the cells through regulation of the dynamics of the actin cytoskeleton, small GTPases of the Rac family, and their guanine nucleotide exchange factors ELMO and Dock180 (Brugnera et al., 2002; Gumienny et al., 2001; Park et al., 2007). A number of other members of the small GTPase family, involved in regulation of the reorganization of the cytoskeleton, were found to affect the engulfment of extraneous objects by macrophages in vitro (Nakaya et al., 2006). Increased activity of some small GTPases enhanced phagocytosis (RhoG, Rac1 and Rab5), RhoA appeared to have an inhibitory effect. In a much simpler and less genetically redundant organism, Dictyostelium discoideum, often used as a basic model of phagocytosis, requirement for the Class VII unconventional myosin for phagocytosis was demonstrated using a genetic approach (Titus, 1999). Recent elegant studies utilizing a number of transgenic mouse tools demonstrated that macrophages, at least in some instances, are also responsible for initiation of cell death, and may serve as an indispensable source of the crucial molecular signals triggering apoptosis. It was discovered that macrophages associated with hyaloid vessels in the developing eye induce PCD in endothelial cells of these vessels by producing a locally acting paracrine signal, Wnt7b, signaling via what is known as the canonical WNT pathway (Lobov et al., 2005). It has been proposed that macrophages secrete Wnt7b in response to angiopoietin 2, which simultaneously suppresses survival of the endothelial cells (Rao et al., 2007). Production of a locally acting factor required to trigger apoptosis by macrophages might serve as an important mechanism ensuring their proximity to the site of the PCD, thus guaranteeing prompt removal of the generated cell debris (Rao et al., 2007). It is difficult to address questions on the global importance of macrophages in the clearance of apoptotic and necrotic cells in vertebrates at this time because no vertebrate model lacking embryonic phagocytes altogether exists, hinting at potential redundancy of the genetic regulation pathways regulating such a pivotal system. Out of the above-mentioned knockouts of the mouse genes involved in the regulation of phagocytosis by macrophages, only inactivation of the potential PS receptor gene, psr, results in developmental abnormalities, and even in that case of a limited and genetic background-dependent nature (Li et al., 2003). The situation is also complicated by the suggested ability of mesenchymal cells to perform, albeit not very efficiently, phagocytic functions in the mouse embryo (Wood et al., 2000). It has to be noted, however, that this observation was made in PU.1-null embryos which, while lacking some of the ‘‘definitive’’, PU.1-dependent macrophages (as suggested by loss of expression of ABC1 and C1q genes), still retain a significant embryonic phagocyte population, expressing, for instance, the CSF-1 receptor gene, Csf1r (Lichanska et al., 1999). Enucleation of the forming definitive erythrocytes is one of the examples of the essential scavenging function

MACROPHAGES IN THE EMBRYO AND BEYOND

performed by macrophages in the embryo. Engulfment of the nuclei ejected from the erythroid precursor cells appears to be dependent on detection by macrophages of the PS exposed on the membrane enveloping the expunged nuclei (Yoshida et al., 2005). Interestingly, impaired macrophage function leading to their inability to interact with erythroblasts and assist in enucleation appears to be an underlying cause of the embryonic lethality of mice homozygous for a null mutation in the retinoblastoma (Rb) gene (Iavarone et al., 2004).

TROPHIC ROLE OF MACROPHAGES The first speculations that macrophages may play a trophic role in tissues of their residence stemmed from recognition of their ability to produce a wide array of secreted factors (Unanue, 1976), and the observations of their abundance and morphological appearance in tissues, facilitated by the use of an antibody against an exquisitely macrophage-specific surface marker, the F4/ 80(EMR1) protein (Gordon et al., 1986). A typical tissue macrophage has a characteristic ramified appearance because of its extensively branching and numerous processes, bolstering multiple interactions with surrounding cells, and increasing the macrophage’s surface. Soon after these original observations, it was demonstrated that macrophages express an impressive cocktail of cytokines and mitogenic factors, including IGF-1, TGF-a, PDGF-A and TGF-b (Rappolee and Werb, 1988), and many other diffusible molecules, including multiple interleukins (ILs), VEGFs, RANKL and LIF (Hume et al., 2002). Brain macrophages (microglia) were also found to be a source of the potent neuron survival and growth factor, the nerve growth factor (Houlgatte et al., 1989; Mallat et al., 1989), and a number of related factors of the neurotrophin family, such as the brain-derived neurotrophic factor and neurotrophin-3 (Elkabes et al., 1996). Mouse models with depleted macrophage populations provided further functional evidence for their important trophic function. It was discovered, for instance, in osteopetrotic(op) homozygous mutant mice lacking the key macrophage growth factor, CSF-1, that recruitment of macrophages, earlier implicated in mammary gland development (Gouon-Evans et al., 2000; Pollard and Hennighausen, 1994), into the gland was essential for its normal development, in particular for ductal branching morphogenesis (Van Nguyen and Pollard, 2002). Regulated re-expression of CSF-1 in the mammary gland epithelium on the mutant op/op background was found to be sufficient to lead to restoration of the macrophage population in the gland and rescue of the normal ductal branching and outgrowth (Van Nguyen and Pollard, 2002). Brain development in the op/op mice also appears to be impaired, and can be largely rescued by administration of CSF-1injection in postnatal mutant mice (Cecchini et al., 1994; Michaelson et al., 1996). The survival and process outgrowth of the neurons taken from adult mice, when brain is already fully devel-

453

oped, however, could be only stimulated by CSF-1 in case of wild type, but not op/op mutant neuronal donors. This suggests that microglia/brain macrophages, thought to be the only cell types expressing the receptor for CSF-1 in the above-mentioned explants, are mediating this trophic effect of CSF-1 (Michaelson et al., 1996). Macrophages also appear to play a vital role in development of the pancreas (in mice and humans), particularly in the formation of the insulin-producing cell population, which is significantly diminished in op homozygous mutant mice lacking CSF-1 (Banaei-Bouchareb et al., 2004, 2006; Geutskens et al., 2005). In the lungs, macrophages were identified as a source of the potent growth factor, IGF-1, playing a crucial role in the survival of myofibroblasts and in the development of idiopathic pulmonary fibrosis (Wynes and Riches, 2003; Wynes et al., 2004). Macrophages also emerged as an important accessory cell type in the maintenance of reproductive function, as highlighted by the impaired fertility of the mutants for both CSF-1 and its receptor, CSF1R (Dai et al., 2002; Pollard, 1997). CSF-1-deficient males have significantly decreased sperm counts and libido due to a low level of synthesized testosterone, while mutant females have low ovulation rates and abnormally long estrous cycles (Cohen et al., 1996, 1999). Interestingly, many of those defects appear to stem from the disregulation of hypothalamic-pituitary interactions, attributed to the defects in microglial function during development (Cohen et al., 1999). In the testis, macrophages make up to 25% of the interstitial cell population, and are well known to establish close intercellular contacts of a very specialized type with steroidogenic Leydig cells in both in vivo and in vitro conditions (Giannessi et al., 2005; Hutson, 1992; Niemi et al., 1986). Similar to the testis, in the ovary macrophages are present in the interstitial tissue and are recruited to the theca layers just before ovulation, probably by the CSF-1 produced by the granulosa cells (Cohen et al., 1997). Macrophages that are recruited to the follicle are thought to play a role in the rupture of the follicle, and possibly in the preluteal repair of the ovarian wall (Brannstrom et al., 1993). The trophic function of embryonic macrophages, with particular emphasis on their role in the modulation of renal development, was recently investigated using a combination of both functional and descriptive approaches (Rae et al., 2007). Similarly to the abovementioned studies, treatment of fetal kidney explants with CSF-1 (thought to act on residential macrophage population, and combined with low doses of lipopolysaccharide) enhanced outgrowth and branching of the ureteric tree (Rae et al., 2007). Macrophages from a few tissues of the mid-gestation embryo were sorted using Csf1r-EGFP reporter fluorescence (Table 1) from other cell types in the source (kidney, lung or brain), and the expression profiles were compared both between the macrophage populations from different tissues and between the macrophages and their source tissue. This profiling revealed a remarkable similarity among the

454

OVCHINNIKOV

populations of residential macrophages from different tissues, as well as the significant overlap between the sets of genes expressed by residential macrophages and the macrophages associated with tumors and considered pro-proliferative, so-called M2 macrophages (Mantovani et al., 2004). The latter macrophage population will be discussed further below.

MACROPHAGES IN REGENERATION AND REPAIR In addition to playing a trophic role in tissues and being a source of a whole cohort of diffusible factors, macrophages from both resident and recruited pools have long been implicated in the repair of damaged tissues and wound healing. In a classical model of wound repair, the healing of the skin wound, initial observations suggested a requirement for wound-recruited macrophages for its normal healing. A depletion of macrophages using a combination of systemic steroids and locally administered macrophage antisera impaired debridement of the wound, i.e., the clearance of dead cells, the fibrin clot, and tissue debris (Leibovich and Ross, 1975). Interestingly, PU.1-deficient neonatal mice, lacking most of the myeloid lineage altogether, display speedier skin wound repair and reduced scarring, and, to further the similarity with embryonic wound healing, is not accompanied by an inflammatory response (Martin et al., 2003). A defect in debridement is not observed in this case probably due to the absence of neutrophilic granulocytes, constituting the major cell population undergoing apoptosis in the wound milieu (Martin and Leibovich, 2005). The dual role of macrophages in healing was highlighted recently in a detailed study using temporally controlled macrophage ablation in a model of inflammation-associated fibrosis in the liver. This study showed that while macrophages contribute to scarring at the early stages, they are required for an efficient scar resolution later on (Duffield et al., 2005). A vast array of genes, including those expressed in macrophages, was identified recently using a microarray-based comparison of the skin wound models from the PU.1-null (macrophage-deficient) and normal mice (Cooper et al., 2005). This approach allowed identification of two distinct gene sets, one associated with the tissue repair itself, and another with the ensuing inflammatory response (Cooper et al., 2005). Macrophages appear to possess a great potential in the regeneration of neural tissue, which does not come as a great surprise considering the spectrum of potent neurotrophic and other factors expressed by the brainassociated macrophage population, microglia. As just one of many examples of the macrophage role in neuroregeneration, implantation of the autologous macrophages was found to greatly promote regeneration of spinal cord injury, resulting in a partial recovery of paraplegic rats (Rapalino et al., 1998). Studies based on this approach culminated in the development of the therapeutic approaches currently in trial on human patients (Knoller et al., 2005; Schwartz et al., 1999). The neuro-

protective role of microglia, found to express IGF-I in addition to nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, was clearly demonstrated in an in vivo inducible ablation experiment in an induced cerebral ischemia setting (Lalancette-Hebert et al., 2007). An increase in the area of infarction was observed after depletion of the microglia, while stimulation of microglial proliferation by addition of CSF-1 had the opposite effect. Macrophage infiltration of the kidney has long been recognized as a hallmark of kidney injury, but it is only recently that a protective, rather than destructive, role of some macrophage subsets has been appreciated (Wilson et al., 2004). It was discovered that in many settings, for instance in ischemia/reperfusion injury, regeneration of the kidney was macrophage-dependent, and was accompanied by the switch of the macrophage state from pro- into anti-inflammatory, followed by production of angiogenic and anti-inflammatory factors such as TGFb and IL-10, in addition to the clearance of cell and tissue debris by macrophages (Ferenbach et al., 2007; Vinuesa et al., 2008). In muscle repair, including the highly clinically important healing after myocardial infarction, the role of macrophages has, until recently, remained largely obscure. Macrophage involvement in itself, however, was clearly demonstrated, for instance, in healing canine infarcts, where locally induced endogenous expression of CSF-1 is accompanied by accumulation and proliferation of a significant macrophage population (Frangogiannis et al., 2003). It was accepted that macrophages recruited to the site of injury are probably responsible for the formation of granulation tissue on one hand, and for scar resolution and the clearance of cell and tissue debris on the other (Frangogiannis, 2006). Recent transplantation experiments using genetically labeled mouse cells indicate that macrophages may still be the ‘‘good guys’’ in healing of the myocardial infarction. It was shown that many of the myofibroblasts forming at the site of the infarction and important in its healing are actually derived from circulating blood monocytes, recruited to the infarction site by trophic factors for monocytes and macrophages, CSF-1 (M-CSF), or GM-CSF, thought to act on some macrophage populations and neutrophilic granulocytes (Fujita et al., 2007). Further studies utilizing transgenic mice uncovered that the recruitment of monocytes is an essential part of successful healing in skeletal muscle in general, which was found to be severely compromised after an inducible monocyte ablation, and is accompanied by a change of the monocytes and macrophages from the inflammatory/nonproliferative into an anti-inflammatory/proliferating state (Arnold et al., 2007; Sonnet et al., 2006).

MACROPHAGES AND TUMORIGENESIS In recent years, the connection between chronic inflammation and cancer has been firmly established (Balkwill

MACROPHAGES IN THE EMBRYO AND BEYOND

and Mantovani, 2001; Balkwill et al., 2005; Coussens and Werb, 2002). A number of studies demonstrated the pivotal role macrophages could play in tumor progression in mice, and described a strong positive correlation between the number of tumor-associated macrophages (TAMs) and tumor invasiveness with poor prognosis for some human cancers (Bingle et al., 2002; Lin et al., 2002, 2001; Pollard, 2004). The involvement of macrophages in tumorigenesis may be traced back as far as the initial transforming events, with inflammatory macrophages being a major source of potently mutagenic reactive oxygen and nitrogen species (Maeda and Akaike, 1998). In addition, a number of inflammatory cytokines produced by macrophages, such as TNFa and MIF, were found to increase the ‘‘transformability’’ of various cell types, while TNFadeficient mice showed lower susceptibility to certain types of cancer (Arnott et al., 2002; Hudson et al., 1999; Moore et al., 1999). Manipulations of gene function in mice highlighted a role in tumorigenesis for the NF-jB pathway, previously primarily recognized as a key pathway in the regulation of inflammation (Greten et al., 2004; Karin and Greten, 2005). TAMs are thought to be recruited to many tumors due to the ability of the latter to express a repertoire of chemokines and growth factors, with notable examples of CCL2 and CSF-1, acting as both chemoattractants and trophic factors (Condeelis and Pollard, 2006; Pollard, 2004). Analysis of certain cancer models in mice confirmed that the absence of macrophages (as is largely the case in CSF-1-deficient mice) greatly reduced the rate of tumor progression and nearly completely prevented the tumor’s metastasis, while elevated levels of CSF-1 in normal mice accelerated tumor progression and augmented its metastatic potential (Condeelis and Pollard, 2006; Lin et al., 2001). Tumor macrophages appear to play an indispensable role in the maintenance and propagation of malignancy. Recruited to hypoxic sites in tumors, macrophages and their monocytic precursors are crucial for angiogenesis in those locales, in large part due to their ability to produce angiogenic factors, notably including VEGF. Multiple lines of evidence point to TAMs as the cells responsible for ‘‘flipping the tumor’s angiogenic switch,’’ and the importance of this lineage in the formation of tumor vasculature was demonstrated by a marked reduction in tumor angiogenesis, and consequent tumor size reduction, resulting from the ablation of proangiogenic monocyte populations (Condeelis and Pollard, 2006; De Palma et al., 2005; Lin and Pollard, 2007). Tumors also hijack the matrix remodeling capabilities of TAMs, commonly found at the invasive front of tumors at various stages, largely mediated by their ability to secrete matrix metalloproteases, such as MMP-9 (Condeelis and Pollard, 2006). TAMs also serve as essential partners for tumor cells in tissue invasion and intravasation, starting with the convergence of the two participating cells types, dependent on reciprocal signaling by EGF produced by TAMs and CSF-1

455

secreted by tumor cells (Condeelis et al., 2005; Wang et al., 2005). Established neoplasias appear to harness the immunomodulatory functions of macrophages, with most TAMs skewed towards the immunosuppressive end of the spectrum, in what is often referred to as an M2 polarization (Biswas et al., 2006, 2008). These M2-polarized TAMs secrete anti-inflammatory cytokines, e.g., IL-10, and not only fail to elicit and sustain an immune response to tumor cells, but actually suppress the ability of other immune cells, such as natural killer (NK) and T cells, to do so (Mantovani et al., 2005; Zitvogel et al., 2006).

SIMILARITIES BETWEEN EMBRYONIC AND TUMOR-ASSOCIATED MACROPHAGES Recently performed microarray-based transcriptional profiling revealed significant similarities between resident macrophages in the embryonic tissues and typical tumor-associated macrophages (TAMs) (Rae et al., 2007). Both cell types are thought to be similar to what is known as the M2, or alternatively activated, macrophages, which suppress inflammation, actively scavenge debris and secrete a wide array of growth factors (Allavena et al., 2007). Consistent with these proposed functions, M2 macrophages are characterized by expression of anti-inflammatory cytokines such as IL-10 and TGFbs, as well as molecules antagonizing the action of proinflammatory cytokines, such as antagonist for the IL-1 receptor (IL-1R) and decoy IL-1RII (Allavena et al., 2007). M2 macrophages, in contrast with the M1, or classically activated macrophages, are very poor antigen presenters, produce very low if any cytotoxic reactive oxygen and nitrogen intermediates, and are normally associated with wound healing, angiogenesis, and tissue remodeling (Allavena et al., 2007). Both embryonic resident and TAM also express high levels of the macrophage mannose receptor (Rae et al., 2007; Sica and Bronte, 2007), another M2 macrophage marker, thought to be involved in recognition of the extracellular determinants on a wide range of pathogens, from viruses to parasitic worms, and possibly of some endogenous mucins (Allavena et al., 2004; Taylor et al., 2005). Macrophage scavenger receptor genes (Msra, Msr2), are also expressed at high levels in both embryonic and TAM, Table 2 (Rae et al., 2007). Both cell populations express a set of potent chemokines of the Ccl and Cxcl families, most prominently Ccl2 and Cxcl2; many other members of these families, e.g., Ccl4, Ccl6, Ccl9, Ccl12, are highly expressed in the tissue embryonic macrophage population as shown in Supporting Information Table 1 (Rae et al., 2007; Sica et al., 2007; Ueno et al., 2000). MACROPHAGES: THE FUSION In a few recent years, the ability of macrophages to participate in cell fusion in various settings has received

456

OVCHINNIKOV

well-deserved attention. It has been known for a long time that monocyte/macrophage lineage cells fuse homotypically (i.e., with each other) during formation of the multinucleated bone-resorbing cells, osteoclasts, and so-called foreign body giant cells, often formed in chronic inflammatory settings (Vignery, 2005b; Yagi et al., 2005). Recently, involvement of the three receptor-ligand pairs in mediating this homotypic macrophage fusion has been firmly established. The first of them consists of the macrophage fusion receptor (MFR, also known as SIRPa), expressed exclusively by the myeloid lineage cells and some neurons, and its ubiquitously expressed ligand CD47, the ‘‘self’’ antigen, both belonging to the immunoglobulin superfamily (Vignery, 2005a). The second pair consists of the novel seventransmembrane-spanning receptor dendritic cell-specific transmembrane protein (DC-STAMP), the requirement for which in multinucleated osteoclast formation has been clearly demonstrated by generation of a knockout mouse, and its unidentified ligand (Yagi et al., 2005). The most recently identified player is the CD200-CD200 receptor pair, bearing a number of similarities to the MFR/SIRPa-CD47 duo, since all four proteins belong to the immunoglobulin superfamily, and expression of both receptors is myeloid lineage-restricted, while both ligands are widely expressed (Cui et al., 2007). (Camargo et al., 2003, 2004a,b). Macrophages (or related cells, such as monocytes or myeloid precursors) were found to be able partners in fusion with a wide array of cell types in many different tissues (Camargo et al., 2004a; Vignery, 2005a). Interestingly, this heterotypic fusion phenomenon is thought to be responsible for what was perceived as the transdifferentiation capacity of the hematopoietic stem cells in transplantation experiments (Camargo et al., 2004a). The liver is probably one of the most extensively studied organs in which macrophages are clearly capable of fusion with resident cells, mostly hepatocytes (Camargo et al., 2004b; Vassilopoulos et al., 2003; Wang et al., 2003). It would even appear that macrophages and their precursors can be successfully used for the cell-based therapies aimed at liver regeneration and amelioration of metabolic disorders (Willenbring et al., 2004). Elegant experiments utilizing a Cre reporter-based lineage tracing approach demonstrated that cells of hematopoietic origin can indeed fuse with, in addition to hepatocytes, neurons and muscle cells, including cardiomyocytes, while no evidence of transdifferentiation of hematopoietic precursors without fusion was uncovered (Alvarez-Dolado et al., 2003; Doyonnas et al., 2004). This is in interesting contrast to the recent finding that circulating myeloid/macrophage precursors can directly give rise to the vascular endothelial cells in a process not involving cell–cell fusion (Bailey et al., 2006). Currently notably lacking, understanding of the molecular mechanisms underlying ability of macrophages to fuse with other cell types might have important implications in development of novel cell-based therapies.

MANIPULATING MACROPHAGE FUNCTION Somewhat surprisingly, a traditional genetic approach consisting of analysis and generation of mutants has so far not proven to be as fruitful in the analysis of macrophage function in mice. Mutants for genes thought to be indispensable for macrophage lineage development in general, including the PU.1 transcription factor, main macrophage trophic factor CSF-1(M-CSF) and its receptor, Csf1r, while providing very useful insights into macrophage function in some organs, unexpectedly appeared to retain multiple subsets of embryonic and adult macrophages (Dai et al., 2002; McKercher et al., 1996; Witmer-Pack et al., 1993). The macrophage lineage in particular, and the myeloid lineage in general, appear to possess significant inherent plasticity, and their development must be controlled by significantly redundant mechanisms. This fact is highlighted by the observation that mice lacking all three main trophic factors, granulocyte-, granulocyte-macrophage, and macrophage -colony stimulating factors (G-CSF, GM-CSF, MCSF/CSF-1) still form macrophages and other myeloid cells (Hibbs et al., 2007). Taken together with the ability of macrophages to originate from either circulating monocytes or, in some instances, local proliferation of the macrophage population, this complicates an accurate assessment of the roles this cell type plays in many organs and tissues. An alternative approach, based on an inducible ablation of macrophages and myeloid cells, will be discussed below. Recent advancements in the field of mouse molecular genetics provided researchers with a number of powerful tools for manipulating gene function in the cell type of choice. A commonly utilized approach to gene inactivation using the Cre-loxP system in macrophages has so far largely relied upon use of the myeloid- rather than macrophage-specific Cre knock-in into the lysozymeM locus (Clausen et al., 1999). Although this Cre line appears highly efficient in neutrophilic granulocytes in vivo (>95% recombination, depending on the allele), its efficiency in the macrophage lineage is significantly lower in an in vivo setting. Another Cre knock-in allele has been reported, in which recombinase is expressed from the locus encoding the definitive macrophage-specific protein marker F4/80(EMR1) (Schaller et al., 2002). It is not clear, however, if Cre expression is confined to the monocyte/macrophage lineage in this case either, since while the F4/80(EMR1) protein is highly macrophage-restricted, the promoter of that gene is also active in granulocytes (Sasmono et al., 2007). A transgenic mouse expressing Cre from the pan-myeloid promoter from the gene encoding definitive myeloid marker Mac1(CD11b), as expected, allows for efficient recombination in all myeloid lineage cells, including granulocytes, macrophages and osteoclasts (Ferron and Vacher, 2005). So far, only one mouse line has been described that could be utilized for gene overexpression, and it is achieved using the Gal4/UAS system, utilizing a modified Csf1r promoter (Ovchinnikov et al., 2008). Since expres-

MACROPHAGES IN THE EMBRYO AND BEYOND

457

sion of the genes of interest from the Gal4-dependent promoter appears to be confined to monocytes, macrophages, and dendritic cells, which are simultaneously labeled by expression of the fluorescent Gal4 reporter, ECFP, this mouse line was called MacBlue. Ablation of the cell type of interest, especially in an inducible fashion, is a powerful approach to addressing the role of this cell population in both development and adult life. Three different transgenic mice were developed, utilizing different molecular mechanisms to achieve inducible depletion of the macrophages and other cells of the myeloid lineage. Ablation of macrophages, and to a lesser extent other cells of the myeloid lineage, in the first published mouse line is achieved by expression of the human diphtheria toxin receptor, targeted to these cells using the CD11b/Mac1 myeloid-specific promoter, and conferring sensitivity to the toxin, which is capable of killing receptive cells even at very low concentrations (Duffield et al., 2005). Interestingly, macrophages in these mice appear to be much more sensitive to diphtheria toxin administration than neutrophilic granulocytes, allowing for their preferential ablation in experimental conditions. Another mouse model used the same promoter to drive expression of the herpes simplex virus thymidine kinase, conferring sensitivity to gancyclovir and its analogues (Gowing et al., 2006), and was largely used so far to study the function of brain macrophages, microglia (Lalancette-Hebert et al., 2007). The third transgenic mouse line permits macrophage Fas-induced apoptosis, and has thus received the name MAFIA (Burnett et al., 2004, 2006). MAFIA mice express a chimaeric Fas apoptosis-inducing receptor, the dimerization and activation of which is dependent on a synthetic compound, under the control of Csf1r promoter. A transgene for regulated ablation has also been used to assess the role macrophages play during development of the clawed frog, Xenopus laevis. The promoter of the gene encoding a well-characterized embryonic leukocyte marker in Xenopus, XLURP-1 (for Xenopus Ly-6/uPARrelated protein), was used to drive expression of a cytotoxic ion channel from the influenza-A virus (Smith et al., 2002, 2007). Its toxicity can be effectively modulated by rimantadine, thus allowing for a controlled cell ablation in this aquatic organism by simple withdrawal of the inhibitor. This approach has been used to illustrate the requirement for macrophages in the frog’s developing embryo (Smith et al., 2007).

‘‘primitive’’ yolk sac-derived and ‘‘definitive’’, derived form the embryo proper, embryonic macrophages. Hopefully, genes regulating development of the former lineage will be identified, as well as transgenic tools generated allowing us to distinguish and differentially manipulate the two populations. Aside from the traditionally recognized essential role as scavengers and cells of the innate and acquired immune systems, macrophages take on many other roles, often related to their ability to express a wide array of secreted factors, and provide trophic support to many important cell types. Macrophages and their precursors also function as very capable fusion partners, fusing with each other or diverse other cell types. This later type of fusion could be partially responsible for the important role macrophages play in tissue repair, and potentially for their contribution to the metastasis of tumors (Vignery, 2005a). Understanding the molecular events leading up and following this heterotypical fusion will probably become one of the very rewarding pursuits of macrophage/myeloid cell biology. This knowledge will have long-reaching implications for our understanding of the cell-cell interaction before and during the fusion, and might provide useful insights for development of a wide range of cell-based therapies. Heterotypical fusion also leads to a unique interaction within the same cell of two (or possibly more) copies of the genome executing genetic programs of very different cell types, and understanding of this interaction will greatly increase our understanding of the epigenetic regulation of genomes of complex organisms. Recently performed comparative microarray profiling of macrophages and neutrophilic granulocytes revealed striking near-identity of the transcriptomes of these two myeloid cell types (Sasmono et al., 2007) and an essential lack of strictly macrophage-specific promoters. This represents a significant methodological caveat for utilizing mouse molecular genetic approaches to study macrophage biology. The most feasible solutions for targeting the expression of proteins of interest (including Cre and Flp recombinases, various forms of the Tet repressor, etc.) to macrophages will include the generation of fusions to proteins known to be translated exclusively in macrophages, such as Mmr, Csf1r, and F4/80 (EMR1). An ability to inactivate gene function, or conversely express gene of interest, efficiently and exclusively in the macrophage lineage will greatly improve our understanding of the biology of this important and influential cell type.

CONCLUDING REMARKS

ACKNOWLEDGMENTS

It has become quite clear in the last decade that macrophages throughout ontogenesis represent one of the most all-pervading and pluripotent cell types. The only tissue free of intercalating macrophages is cartilage masses which are, incidentally, devoid of any vasculature. One of the areas of macrophage biology requiring significant clarification is the relationship between the

The author is grateful to Drs Melissa Little and Kate Schroder for providing access to and assisting with interpretation of the microarray data, respectively (Summarized in Supporting Information Table 1). The author thanks the Genomics Institute of the Novartis Research Foundation SymAtlas (http://symatlas.gnf.org/SymAtlas/) team (and in particular John Walker and Andrew Su) for

458

OVCHINNIKOV

sharing the data from GNF SymAtlas v. 3 dataset prior to its publication and public release. The author also thanks Dr Deanne Whitworth for the helpful comments on the manuscript. LITERATURE CITED Allavena P, Chieppa M, Monti P, Piemonti L. 2004. From pattern recognition receptor to regulator of homeostasis: The double-faced macrophage mannose receptor. Crit Rev Immunol 24:179–192. Allavena P, Sica A, Solinas G, Porta C, Mantovani A. 2008. The inflammatory micro-environment in tumor progression: The role of tumorassociated macrophages. Crit Rev Oncol Hematol 66:1–9. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A. 2003. Fusion of bonemarrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425:968–973. Appelt U, Sheriff A, Gaipl US, Kalden JR, Voll RE, Herrmann M. 2005. Viable, apoptotic and necrotic monocytes expose phosphatidylserine: Cooperative binding of the ligand Annexin V to dying but not viable cells and implications for PS-dependent clearance. Cell Death Differ 12:194–196. Arnold L, Henry A, Poron F, Baba-Amer Y, van Rooijen N, Plonquet A, Gherardi RK, Chazaud B. 2007. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J Exp Med 204:1057–1069. Arnott CH, Scott KA, Moore RJ, Hewer A, Phillips DH, Parker P, Balkwill FR, Owens DM. 2002. Tumour necrosis factor-alpha mediates tumour promotion via a PKC alpha- and AP-1-dependent pathway. Oncogene 21:4728–4738. Aschoff L. 1924. Das reticulo-endotheliale system. Ergebnisse der Inneren Medicine und Kinderheilkinde 26:1–118. Asha H, Nagy I, Kovacs G, Stetson D, Ando I, Dearolf CR. 2003. Analysis of Ras-induced overproliferation in Drosophila hemocytes. Genetics 163:203–215. Bailey AS, Willenbring H, Jiang S, Anderson DA, Schroeder DA, Wong MH, Grompe M, Fleming WH. 2006. Myeloid lineage progenitors give rise to vascular endothelium. Proc Natl Acad Sci USA 103:13156–13161. Balkwill F, Charles KA, Mantovani A. 2005. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7:211–217. Balkwill F, Mantovani A. 2001. Inflammation and cancer: Back to Virchow? Lancet 357:539–545. Banaei-Bouchareb L, Gouon-Evans V, Samara-Boustani D, Castellotti MC, Czernichow P, Pollard JW, Polak M. 2004. Insulin cell mass is altered in Csf1op/Csf1op macrophage-deficient mice. J Leukoc Biol 76:359–367. Banaei-Bouchareb L, Peuchmaur M, Czernichow P, Polak M. 2006. A transient microenvironment loaded mainly with macrophages in the early developing human pancreas. J Endocrinol 188:467–480. Bellen HJ, O’Kane CJ, Wilson C, Grossniklaus U, Pearson RK, Gehring WJ. 1989. P-element-mediated enhancer detection: A versatile method to study development in Drosophila. Genes Dev 3:1288–1300. Bernardoni R, Vivancos V, Giangrande A. 1997. Glide/gcm is expressed and required in the scavenger cell lineage. Dev Biol 191:118–130. Bertrand JY, Jalil A, Klaine M, Jung S, Cumano A, Godin I. 2005. Three pathways to mature macrophages in the early mouse yolk sac. Blood 106:3004–3011. Bingle L, Brown NJ, Lewis CE. 2002. The role of tumour-associated macrophages in tumour progression: Implications for new anticancer therapies. J Pathol 196:254–265. Biswas SK, Gangi L, Paul S, Schioppa T, Saccani A, Sironi M, Bottazzi B, Doni A, Vincenzo B, Pasqualini F, Vago L, Nebuloni M, Mantovani A, Sica A. 2006. A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood 107:2112–2122. Biswas SK, Sica A, Lewis CE. 2008. Plasticity of macrophage function during tumor progression: Regulation by distinct molecular mechanisms. J Immunol 180:2011–2017.

Bondanza A, Zimmermann VS, Rovere-Querini P, Turnay J, Dumitriu IE, Stach CM, Voll RE, Gaipl US, Bertling W, Poschl E, Kalden JR, Manfredi AA, Herrmann M. 2004. Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo. J Exp Med 200:1157–1165. Bonifer C, Faust N, Geiger H, Muller AM. 1998. Developmental changes in the differentiation capacity of haematopoietic stem cells. Immunol Today 19:236–241. Bose J, Gruber AD, Helming L, Schiebe S, Wegener I, Hafner M, Beales M, Kontgen F, Lengeling A. 2004. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal. J Biol 3:15. Brannstrom M, Mayrhofer G, Robertson SA. 1993. Localization of leukocyte subsets in the rat ovary during the periovulatory period. Biol Reprod 48:277–286. Brown S, Heinisch I, Ross E, Shaw K, Buckley CD, Savill J. 2002. Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature 418:200–203. Brugnera E, Haney L, Grimsley C, Lu M, Walk SF, Tosello-Trampont AC, Macara IG, Madhani H, Fink GR, Ravichandran KS. 2002. Unconventional Rac-GEF activity is mediated through the Dock180ELMO complex. Nat Cell Biol 4:574–582. Burnett SH, Beus BJ, Avdiushko R, Qualls J, Kaplan AM, Cohen DA. 2006. Development of peritoneal adhesions in macrophage depleted mice. J Surg Res 131:296–301. Burnett SH, Kershen EJ, Zhang J, Zeng L, Straley SC, Kaplan AM, Cohen DA. 2004. Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J Leukoc Biol 75:612– 623. Camargo FD, Chambers SM, Goodell MA. 2004a. Stem cell plasticity: From transdifferentiation to macrophage fusion. Cell Prolif 37:55– 65. Camargo FD, Finegold M, Goodell MA. 2004b. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. J Clin Invest 113:1266–1270. Camargo FD, Green R, Capetanaki Y, Jackson KA, Goodell MA. 2003. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 9:1520–1527. Cecchini MG, Dominguez MG, Mocci S, Wetterwald A, Felix R, Fleisch H, Chisholm O, Hofstetter W, Pollard JW, Stanley ER. 1994. Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development 120:1357–1372. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. 1999. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 8:265–277. Cohen PE, Chisholm O, Arceci RJ, Stanley ER, Pollard JW. 1996. Absence of colony-stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice results in male fertility defects. Biol Reprod 55:310–317. Cohen PE, Nishimura K, Zhu L, Pollard JW. 1999. Macrophages: Important accessory cells for reproductive function. J Leukoc Biol 66:765–772. Cohen PE, Zhu L, Pollard JW. 1997. Absence of colony stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice disrupts estrous cycles and ovulation. Biol Reprod 56:110–118. Condeelis J, Pollard JW. 2006. Macrophages: Obligate partners for tumor cell migration, invasion, and metastasis. Cell 124:263–266. Condeelis J, Singer RH, Segall JE. 2005. The great escape: When cancer cells hijack the genes for chemotaxis and motility. Annu Rev Cell Dev Biol 21:695–718. Cooper L, Johnson C, Burslem F, Martin P. 2005. Wound healing and inflammation genes revealed by array analysis of ‘macrophageless’ PU.1 null mice. Genome Biol 6:R5. Coussens LM, Werb Z. 2002. Inflammation and cancer. Nature 420:860–867. Cui W, Cuartas E, Ke J, Zhang Q, Einarsson HB, Sedgwick JD, Li J, Vignery A. 2007. CD200 and its receptor, CD200R, modulate bone mass via the differentiation of osteoclasts. Proc Natl Acad Sci USA 104:14436–14441. Cumano A, Dieterlen-Lievre F, Godin I. 1996. Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86:907–916.

MACROPHAGES IN THE EMBRYO AND BEYOND

Cumano A, Ferraz JC, Klaine M, Di Santo JP, Godin I. 2001. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution. Immunity 15:477–485. Cumano A, Godin I. 2007. Ontogeny of the hematopoietic system. Annu Rev Immunol 25:745–785. Dai XM, Ryan GR, Hapel AJ, Dominguez MG, Russell RG, Kapp S, Sylvestre V, Stanley ER. 2002. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood 99:111– 120. De Palma M, Venneri MA, Galli R, Sergi Sergi L, Politi LS, Sampaolesi M, Naldini L. 2005. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 8:211–226. Dieterlen-Lievre F. 1975. On the origin of haemopoietic stem cells in the avian embryo: An experimental approach. J Embryol Exp Morphol 33:607–619. Doyonnas R, LaBarge MA, Sacco A, Charlton C, Blau HM. 2004. Hematopoietic contribution to skeletal muscle regeneration by myelomonocytic precursors. Proc Natl Acad Sci USA 101:13507– 13512. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 115:56–65. Dzierzak E, Medvinsky A, de Bruijn M. 1998. Qualitative and quantitative aspects of haematopoietic cell development in the mammalian embryo. Immunol Today 19:228–236. Elkabes S, DiCicco-Bloom EM, Black IB. 1996. Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function. J Neurosci 16:2508–2521. Ellis RE, Jacobson DM, Horvitz HR. 1991. Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans. Genetics 129:79–94. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207–2216. Faust N, Huber MC, Sippel AE, Bonifer C. 1997. Different macrophage populations develop from embryonic/fetal and adult hematopoietic tissues. Exp Hematol 25:432–444. Ferenbach D, Kluth DC, Hughes J. 2007. Inflammatory cells in renal injury and repair. Semin Nephrol 27:250–259. Ferron M, Vacher J. 2005. Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice. Genesis 41:138– 145. Franc NC, Dimarcq JL, Lagueux M, Hoffmann J, Ezekowitz RA. 1996. Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 4:431–443. Frangogiannis NG. 2006. Targeting the inflammatory response in healing myocardial infarcts. Curr Med Chem 13:1877–1893. Frangogiannis NG, Mendoza LH, Ren G, Akrivakis S, Jackson PL, Michael LH, Smith CW, Entman ML. 2003. MCSF expression is induced in healing myocardial infarcts and may regulate monocyte and endothelial cell phenotype. Am J Physiol Heart Circ Physiol 285:H483–H492. Fujita J, Mori M, Kawada H, Ieda Y, Tsuma M, Matsuzaki Y, Kawaguchi H, Yagi T, Yuasa S, Endo J, Hotta T, Ogawa S, Okano H, Yozu R, Ando K, Fukuda K. 2007. Administration of granulocyte colonystimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair. Stem Cells 25:2750–2759. Gaipl US, Munoz LE, Rodel F, Pausch F, Frey B, Brachvogel B, von der Mark K, Poschl E. 2007. Modulation of the immune system by dying cells and the phosphatidylserine-ligand annexin A5. Autoimmunity 40:254–259. Gardai SJ, Bratton DL, Ogden CA, Henson PM. 2006. Recognition ligands on apoptotic cells: A perspective. J Leukoc Biol 79:896– 903.

459

Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, MurphyUllrich JE, Bratton DL, Oldenborg PA, Michalak M, Henson PM. 2005. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123:321–334. Geutskens SB, Otonkoski T, Pulkkinen MA, Drexhage HA, Leenen PJ. 2005. Macrophages in the murine pancreas and their involvement in fetal endocrine development in vitro. J Leukoc Biol 78:845–852. Giannessi F, Giambelluca MA, Scavuzzo MC, Ruffoli R. 2005. Ultrastructure of testicular macrophages in aging mice. J Morphol 263:39– 46. Goebeler M, Roth J, Henseleit U, Sunderkotter C, Sorg C. 1993. Expression and complex assembly of calcium-binding proteins MRP8 and MRP14 during differentiation of murine myelomonocytic cells. J Leukoc Biol 53:11–18. Goicoechea S, Orr AW, Pallero MA, Eggleton P, Murphy-Ullrich JE. 2000. Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin. J Biol Chem 275:36358– 36368. Gordon N. 1995. Apoptosis (programmed cell death) and other reasons for elimination of neurons and axons. Brain Dev 17:73–77. Gordon S, Crocker PR, Morris L, Lee SH, Perry VH, Hume DA. 1986. Localization and function of tissue macrophages. Ciba Found Symp 118:54–67. Gouon-Evans V, Rothenberg ME, Pollard JW. 2000. Postnatal mammary gland development requires macrophages and eosinophils. Development 127:2269–2282. Gowing G, Vallieres L, Julien JP. 2006. Mouse model for ablation of proliferating microglia in acute CNS injuries. Glia 53:331–337. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, Kagnoff MF, Karin M. 2004. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 118:285–296. Gumienny TL, Brugnera E, Tosello-Trampont AC, Kinchen JM, Haney LB, Nishiwaki K, Walk SF, Nemergut ME, Macara IG, Francis R, Schedl T, Qin Y, Van Aelst L, Hengartner MO, Ravichandran KS. 2001. CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration. Cell 107:27–41. Hamon Y, Broccardo C, Chambenoit O, Luciani MF, Toti F, Chaslin S, Freyssinet JM, Devaux PF, McNeish J, Marguet D, Chimini G. 2000. ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. Nat Cell Biol 2:399–406. Hanayama R, Tanaka M, Miwa K, Nagata S. 2004a. Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells. J Immunol 172:3876–3882. Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. 2002. Identification of a factor that links apoptotic cells to phagocytes. Nature 417:182–187. Hanayama R, Tanaka M, Miyasaka K, Aozasa K, Koike M, Uchiyama Y, Nagata S. 2004b. Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science 304:1147–1150. Henson PM, Hume DA. 2006. Apoptotic cell removal in development and tissue homeostasis. Trends Immunol 27:244–250. Herbomel P, Thisse B, Thisse C. 1999. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126:3735– 3745. Herbomel P, Thisse B, Thisse C. 2001. Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process. Dev Biol 238:274–288. Hibbs ML, Quilici C, Kountouri N, Seymour JF, Armes JE, Burgess AW, Dunn AR. 2007. Mice lacking three myeloid colony-stimulating factors (G-CSF, GM-CSF, and M-CSF) still produce macrophages and granulocytes and mount an inflammatory response in a sterile model of peritonitis. J Immunol 178:6435–6443. Holz A, Bossinger B, Strasser T, Janning W, Klapper R. 2003. The two origins of hemocytes in Drosophila. Development 130:4955– 4962. Houlgatte R, Mallat M, Brachet P, Prochiantz A. 1989. Secretion of nerve growth factor in cultures of glial cells and neurons derived from different regions of the mouse brain. J Neurosci Res 24:143–152.

460

OVCHINNIKOV

Hudson JD, Shoaibi MA, Maestro R, Carnero A, Hannon GJ, Beach DH. 1999. A proinflammatory cytokine inhibits p53 tumor suppressor activity. J Exp Med 190:1375–1382. Hume DA. 2006. The mononuclear phagocyte system. Curr Opin Immunol 18:49–53. Hume DA, Gordon S. 1983. Mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80. Identification of resident macrophages in renal medullary and cortical interstitium and the juxtaglomerular complex. J Exp Med 157:1704–1709. Hume DA, Monkley SJ, Wainwright BJ. 1995. Detection of c-fms protooncogene in early mouse embryos by whole mount in situ hybridization indicates roles for macrophages in tissue remodelling. Br J Haematol 90:939–942. Hume DA, Ross IL, Himes SR, Sasmono RT, Wells CA, Ravasi T. 2002. The mononuclear phagocyte system revisited. J Leukoc Biol 72:621–627. Hutson JC. 1992. Development of cytoplasmic digitations between Leydig cells and testicular macrophages of the rat. Cell Tissue Res 267:385–389. Huynh ML, Fadok VA, Henson PM. 2002. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-b1 secretion and the resolution of inflammation. J Clin Invest 109:41–50. Iavarone A, King ER, Dai XM, Leone G, Stanley ER, Lasorella A. 2004. Retinoblastoma promotes definitive erythropoiesis by repressing Id2 in fetal liver macrophages. Nature 432:1040–1045. Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, Littman DR. 2000. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20:4106–4114. Karin M, Greten FR. 2005. NF-jB: Linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5:749– 759. Knoller N, Auerbach G, Fulga V, Zelig G, Attias J, Bakimer R, Marder JB, Yoles E, Belkin M, Schwartz M, Hadani M. 2005. Clinical experience using incubated autologous macrophages as a treatment for complete spinal cord injury: Phase I study results. J Neurosurg Spine 3:173–181. Kodama T, Doi T, Suzuki H, Takahashi K, Wada Y, Gordon S. 1996. Collagenous macrophage scavenger receptors. Curr Opin Lipidol 7:287–291. Kunisaki Y, Masuko S, Noda M, Inayoshi A, Sanui T, Harada M, Sasazuki T, Fukui Y. 2004. Defective fetal liver erythropoiesis and T lymphopoiesis in mice lacking the phosphatidylserine receptor. Blood 103:3362–3364. Lalancette-Hebert M, Gowing G, Simard A, Weng YC, Kriz J. 2007. Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain. J Neurosci 27:2596–2605. Lanot R, Zachary D, Holder F, Meister M. 2001. Postembryonic hematopoiesis in Drosophila. Dev Biol 230:243–257. Leibovich SJ, Ross R. 1975. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am J Pathol 78:71–100. Li MO, Sarkisian MR, Mehal WZ, Rakic P, Flavell RA. 2003. Phosphatidylserine receptor is required for clearance of apoptotic cells. Science 302:1560–1563. Lichanska AM, Browne CM, Henkel GW, Murphy KM, Ostrowski MC, McKercher SR, Maki RA, Hume DA. 1999. Differentiation of the mononuclear phagocyte system during mouse embryogenesis: The role of transcription factor PU. 1. Blood 94:127–138. Lichanska AM, Hume DA. 2000. Origins and functions of phagocytes in the embryo. Exp Hematol 28:601–611. Lin CS, Aebersold RH, Kent SB, Varma M, Leavitt J. 1988. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts. Mol Cell Biol 8:4659– 4668. Lin EY, Gouon-Evans V, Nguyen AV, Pollard JW. 2002. The macrophage growth factor CSF-1 in mammary gland development and tumor progression. J Mammary Gland Biol Neoplasia 7:147–162. Lin EY, Nguyen AV, Russell RG, Pollard JW. 2001. Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med 193:727–740.

Lin EY, Pollard JW. 2007. Tumor-associated macrophages press the angiogenic switch in breast cancer. Cancer Res 67:5064–5066. Lobov IB, Rao S, Carroll TJ, Vallance JE, Ito M, Ondr JK, Kurup S, Glass DA, Patel MS, Shu W, Morrisey EE, McMahon AP, Karsenty G, Lang RA. 2005. WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature. Nature 437:417–421. Luciani MF, Chimini G. 1996. The ATP binding cassette transporter ABC1, is required for the engulfment of corpses generated by apoptotic cell death. EMBO J 15:226–235. Maeda H, Akaike T. 1998. Nitric oxide and oxygen radicals in infection, inflammation, and cancer. Biochemistry (Mosc) 63:854–865. Mallat M, Houlgatte R, Brachet P, Prochiantz A. 1989. Lipopolysaccharide-stimulated rat brain macrophages release NGF in vitro. Dev Biol 133:309–311. Mantovani A, Sica A, Locati M. 2005. Macrophage polarization comes of age. Immunity 23:344–346. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. 2004. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25:677–686. Martin P, D’Souza D, Martin J, Grose R, Cooper L, Maki R, McKercher SR. 2003. Wound healing in the PU. 1 null mouse–tissue repair is not dependent on inflammatory cells. Curr Biol 13:1122– 1128. Martin P, Leibovich SJ. 2005. Inflammatory cells during wound repair: The good, the bad and the ugly. Trends Cell Biol 15:599– 607. McKercher SR, Torbett BE, Anderson KL, Henkel GW, Vestal DJ, Baribault H, Klemsz M, Feeney AJ, Wu GE, Paige CJ, Maki RA. 1996. Targeted disruption of the PU. 1 gene results in multiple hematopoietic abnormalities. EMBO J 15:5647–5658. Medvinsky A, Dzierzak E. 1996. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86:897–906. Meister M, Lagueux M. 2003. Drosophila blood cells. Cell Microbiol 5:573–580. Metchnikoff E. 1893. Lectures on the comparative pathology of inflammation. London: Kegan Paul Trench Tubner. Michaelson MD, Bieri PL, Mehler MF, Xu H, Arezzo JC, Pollard JW, Kessler JA. 1996. CSF-1 deficiency in mice results in abnormal brain development. Development 122:2661–2672. Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. 2007. Identification of Tim4 as a phosphatidylserine receptor. Nature 450:435–439. Moore RJ, Owens DM, Stamp G, Arnott C, Burke F, East N, Holdsworth H, Turner L, Rollins B, Pasparakis M, Kollias G, Balkwill F. 1999. Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis. Nat Med 5:828–831. Morris L, Graham CF, Gordon S. 1991. Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80. Development 112:517–526. Nakano T, Ishimoto Y, Kishino J, Umeda M, Inoue K, Nagata K, Ohashi K, Mizuno K, Arita H. 1997. Cell adhesion to phosphatidylserine mediated by a product of growth arrest-specific gene 6. J Biol Chem 272:29411–29414. Nakaya M, Tanaka M, Okabe Y, Hanayama R, Nagata S. 2006. Opposite effects of rho family GTPases on engulfment of apoptotic cells by macrophages. J Biol Chem 281:8836–8842. Niemi M, Sharpe RM, Brown WR. 1986. Macrophages in the interstitial tissue of the rat testis. Cell Tissue Res 243:337–344. Oldenborg PA, Zheleznyak A, Fang YF, Lagenaur CF, Gresham HD, Lindberg FP. 2000. Role of CD47 as a marker of self on red blood cells. Science 288:2051–2054. Osborne BA. 1998. When cells die: A comprehensive evaluation of apoptosis and programmed cell death. New York: Wiley-Liss. pp 245– 266. Ovchinnikov DA, van Zuylen WJ, DeBats CEE, Alexander KA, Kellie S, Hume DA. 2008. Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labelled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UASECFP double-transgenic mice. J Leukoc Biol 83:430–433. Paladi M, Tepass U. 2004. Function of Rho GTPases in embryonic blood cell migration in Drosophila. J Cell Sci 117:6313–6326.

MACROPHAGES IN THE EMBRYO AND BEYOND

Palaniyar N, Nadesalingam J, Clark H, Shih MJ, Dodds AW, Reid KB. 2004. Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannosebinding lectin. J Biol Chem 279:32728–32736. Palis J, Chan RJ, Koniski A, Patel R, Starr M, Yoder MC. 2001. Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis. Proc Natl Acad Sci USA 98:4528–4533. Park D, Tosello-Trampont AC, Elliott MR, Lu M, Haney LB, Ma Z, Klibanov AL, Mandell JW, Ravichandran KS. 2007. BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/ Dock180/Rac module. Nature 450:430–434. Passey RJ, Williams E, Lichanska AM, Wells C, Hu S, Geczy CL, Little MH, Hume DA. 1999. A null mutation in the inflammation-associated S100 protein S100A8 causes early resorption of the mouse embryo. J Immunol 163:2209–2216. Penaloza C, Lin L, Lockshin RA, Zakeri Z. 2006. Cell death in development: Shaping the embryo. Histochem Cell Biol 126:149–158. Pollard JW. 1997. Role of colony-stimulating factor-1 in reproduction and development. Mol Reprod Dev 46:54–60; discussion 60–51. Pollard JW. 2004. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 4:71–78. Pollard JW, Hennighausen L. 1994. Colony stimulating factor 1 is required for mammary gland development during pregnancy. Proc Natl Acad Sci USA 91:9312–9316. Rae F, Woods K, Sasmono T, Campanale N, Taylor D, Ovchinnikov DA, Grimmond SM, Hume DA, Ricardo SD, Little MH. 2007. Characterisation and trophic functions of murine embryonic macrophages based upon the use of a Csf1r-EGFP transgene reporter. Dev Biol 308:232–246. Ramet M, Pearson A, Manfruelli P, Li X, Koziel H, Gobel V, Chung E, Krieger M, Ezekowitz RA. 2001. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 15:1027– 1038. Rao S, Lobov IB, Vallance JE, Tsujikawa K, Shiojima I, Akunuru S, Walsh K, Benjamin LE, Lang RA. 2007. Obligatory participation of macrophages in an angiopoietin 2-mediated cell death switch. Development 134:4449–4458. Rapalino O, Lazarov-Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M, Solomon A, Gepstein R, Katz A, Belkin M, Hadani M, Schwartz M. 1998. Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nat Med 4:814–821. Rappolee DA, Werb Z. 1988. Secretory products of phagocytes. Curr Opin Immunol 1:47–55. Sasmono RT, Ehrnsperger A, Cronau SL, Ravasi T, Kandane R, Hickey MJ, Cook AD, Himes SR, Hamilton JA, Hume DA. 2007. Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1. J Leukoc Biol 82:111–123. Sasmono RT, Oceandy D, Pollard JW, Tong W, Pavli P, Wainwright BJ, Ostrowski MC, Himes SR, Hume DA. 2003. A macrophage colonystimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101:1155–1163. Savill J, Dransfield I, Gregory C, Haslett C. 2002. A blast from the past: Clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2:965–975. Schaller E, Macfarlane AJ, Rupec RA, Gordon S, McKnight AJ, Pfeffer K. 2002. Inactivation of the F4/80 glycoprotein in the mouse germ line. Mol Cell Biol 22:8035–8043. Schwartz M, Lazarov-Spiegler O, Rapalino O, Agranov I, Velan G, Hadani M. 1999. Potential repair of rat spinal cord injuries using stimulated homologous macrophages. Neurosurgery 44:1041–1045; discussion 1045–1046. Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, Earp HS, Matsushima GK. 2001. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411:207–211. Seitz HM, Camenisch TD, Lemke G, Earp HS, Matsushima GK. 2007. Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in clearance of apoptotic cells. J Immunol 178:5635– 5642.

461

Shepard JL, Zon LI. 2000. Developmental derivation of embryonic and adult macrophages. Curr Opin Hematol 7:3–8. Shinomiya H, Hirata H, Saito S, Yagisawa H, Nakano M. 1994. Identification of the 65-kDa phosphoprotein in murine macrophages as a novel protein: Homology with human L-plastin. Biochem Biophys Res Commun 202:1631–1638. Sica A, Bronte V. 2007. Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest 117: 1155–1166. Sica A, Rubino L, Mancino A, Larghi P, Porta C, Rimoldi M, Solinas G, Locati M, Allavena P, Mantovani A. 2007. Targeting tumour-associated macrophages. Expert Opin Ther Targets 11:1219–1229. Smith SJ, Kotecha S, Towers N, Latinkic BV, Mohun TJ. 2002. XPOX2peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus. Mech Dev 117:173–186. Smith SJ, Kotecha S, Towers N, Mohun TJ. 2007. Targeted cell-ablation in Xenopus embryos using the conditional, toxic viral protein M2(H37A). Dev Dyn 236:2159–2171. Sonnet C, Lafuste P, Arnold L, Brigitte M, Poron F, Authier FJ, Chretien F, Gherardi RK, Chazaud B. 2006. Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems. J Cell Sci 119:2497–2507. Tauber AI. 1991. Metchnikoff and the origins of immunology: From metaphor to theory. New York: Oxford University Press. Tavian M, Hallais MF, Peault B. 1999. Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo. Development 126:793–803. Tavian M, Robin C, Coulombel L, Peault B. 2001. The human embryo, but not its yolk sac, generates lympho-myeloid stem cells: Mapping multipotent hematopoietic cell fate in intraembryonic mesoderm. Immunity 15:487–495. Taylor PR, Gordon S, Martinez-Pomares L. 2005. The mannose receptor: Linking homeostasis and immunity through sugar recognition. Trends Immunol 26:104–110. Tepass U, Fessler LI, Aziz A, Hartenstein V. 1994. Embryonic origin of hemocytes and their relationship to cell death in Drosophila. Development 120:1829–1837. Titus MA. 1999. A class VII unconventional myosin is required for phagocytosis. Curr Biol 9:1297–1303. Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, Koike M, Inadera H, Matsushima K. 2000. Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res 6:3282–3289. Unanue ER. 1976. Secretory function of mononuclear phagocytes: A review. Am J Pathol 83:396–417. van Furth R, Cohn ZA. 1968. The origin and kinetics of mononuclear phagocytes. J Exp Med 128:415–435. Van Nguyen A, Pollard JW. 2002. Colony stimulating factor-1 is required to recruit macrophages into the mammary gland to facilitate mammary ductal outgrowth. Dev Biol 247:11–25. Vassilopoulos G, Wang PR, Russell DW. 2003. Transplanted bone marrow regenerates liver by cell fusion. Nature 422:901–904. Vignery A. 2005a. Macrophage fusion: Are somatic and cancer cells possible partners? Trends Cell Biol 15:188–193. Vignery A. 2005b. Macrophage fusion: The making of osteoclasts and giant cells. J Exp Med 202:337–340. Vinuesa E, Hotter G, Jung M, Herrero-Fresneda I, Torras J, Sola A. 2008. Macrophage involvement in the kidney repair phase after ischaemia/reperfusion injury. J Pathol 214:104–113. Wang W, Goswami S, Sahai E, Wyckoff JB, Segall JE, Condeelis JS. 2005. Tumor cells caught in the act of invading: Their strategy for enhanced cell motility. Trends Cell Biol 15:138–145. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M. 2003. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422:897–901. Willenbring H, Bailey AS, Foster M, Akkari Y, Dorrell C, Olson S, Finegold M, Fleming WH, Grompe M. 2004. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 10:744–748. Williamson P, Schlegel RA. 2004. Hide and seek: the secret identity of the phosphatidylserine receptor. J Biol 3:14.

462

OVCHINNIKOV

Wilson HM, Walbaum D, Rees AJ. 2004. Macrophages and the kidney. Curr Opin Nephrol Hypertens 13:285–290. Witmer-Pack MD, Hughes DA, Schuler G, Lawson L, McWilliam A, Inaba K, Steinman RM, Gordon S. 1993. Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse. J Cell Sci 104 (Part 4):1021–1029. Wood W, Jacinto A. 2007. Drosophila melanogaster embryonic haemocytes: Masters of multitasking. Nat Rev Mol Cell Biol 8:542–551. Wood W, Turmaine M, Weber R, Camp V, Maki RA, McKercher SR, Martin P. 2000. Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU. 1 null mouse embryos. Development 127:5245–5252. Wynes MW, Frankel SK, Riches DW. 2004. IL-4-induced macrophagederived IGF-I protects myofibroblasts from apoptosis following growth factor withdrawal. J Leukoc Biol 76:1019–1027.

Wynes MW, Riches DW. 2003. Induction of macrophage insulin-like growth factor-I expression by the Th2 cytokines IL-4 and IL-13. J Immunol 171:3550–3559. Yagi M, Miyamoto T, Sawatani Y, Iwamoto K, Hosogane N, Fujita N, Morita K, Ninomiya K, Suzuki T, Miyamoto K, Oike Y, Takeya M, Toyama Y, Suda T. 2005. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med 202:345– 351. Yoshida H, Kawane K, Koike M, Mori Y, Uchiyama Y, Nagata S. 2005. Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells. Nature 437: 754–758. Zitvogel L, Tesniere A, Kroemer G. 2006. Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol 6:715–727.