Review

Beyond Stem Cells: Self-Renewal of Differentiated Macrophages

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Science  22 Nov 2013:
Vol. 342, Issue 6161, 1242974
DOI: 10.1126/science.1242974

Structured Abstract

Background

Many mature cells of the body are continuously replaced, particularly in tissues that are most exposed to the environment such as cells of the immune system. The need for new cells is driven by cellular turnover during normal tissue homeostasis and is further increased upon infection. Because differentiated cells typically withdraw from the cell cycle, replacement of mature cells is generally thought to depend on differentiation of self-renewing, tissue-specific stem cells. Until recently, tissue macrophages were thought to follow such a pathway, developing from hematopoietic stem cells via bone marrow–progenitor and blood monocyte intermediates. But this view has changed of late with several observations indicating that macrophages can self-renew by local proliferation of mature differentiated cells.

Embedded Image

Macrophage origin and self-renewal. In the classical view, macrophages develop from self-renewing hematopoietic stem cells (HSC) in the bone marrow (BM) via blood monocyte intermediates. However, new data show that some adult tissue macrophage populations develop from embryonic progenitors independent of HSCs and can self-renew. Local proliferation can assure homeostatic maintenance (dotted arrows) and dramatically increase cell number (solid arrows) upon challenge.

Advances

Recent studies have demonstrated that in macrophages, differentiation and cell cycle withdrawal can be uncoupled by the inactivation of specific transcription factors. These cells can then be expanded indefinitely as functionally differentiated macrophages without tumorigenic transformation. At the same time, it became clear that mature macrophages could also expand massively in vivo in response to infections by local proliferation, independently of input from adult hematopoietic stem cells. Furthermore, several populations of tissue macrophages were found to be derived from embryonic progenitors, and macrophages can be self-maintained in adult tissues by local proliferation. Together, these recent data suggest that macrophages are mature differentiated cells that may be endowed with self-renewal capacity akin to that of stem cells.

Outlook

These findings challenge the classical view of tissue maintenance by adult tissue-specific stem cells and indicate that stem cell–like self-renewal mechanisms may be activated in mature differentiated cells. It will be important to determine whether the engaged pathways resemble those active in stem cells and whether they might be activated in other cell types as well. Furthermore, we need to understand how such self-renewal capacity differs from uncontrolled proliferation induced by oncogenic transformation. A first step will be to explore how macrophage proliferation is regulated in vivo: How do macrophages adapt their cell numbers to diverse tissue requirements, from near quiescence during homeostasis to massive expansion under challenge? Macrophages are present in nearly every tissue and serve important functions in immunity, cancer, metabolism, and tissue repair. The role of local macrophage proliferation in these processes has remained largely unexplored. It will be important to investigate how the consequences of macrophage accumulation by local proliferation differ from those of monocyte-derived macrophage recruitment under inflammatory conditions. The control of macrophage numbers independent of inflammatory signals may provide new opportunities for therapeutic intervention in many of these areas.

Macrophage Makeover

Macrophages are important immune cells that function in tissue repair during homeostasis and in the innate immune response. Inflammation, which can be triggered by infection, is accompanied by a massive expansion of macrophages in affected tissues. The major source of this increase in resident macrophages has been thought to be hematopoietic stem cells in the bone marrow. However, recent results have shown that the mature differentiated macrophages residing in the affected tissues can themselves proliferate to boost cell numbers. Sieweke and Allen (10.1126/science.1242974) review what we know about the origin of macrophages and outline the consequences of local macrophage proliferation for the immune response and tissue homeostasis.

Abstract

In many mammalian tissues, mature differentiated cells are replaced by self-renewing stem cells, either continuously during homeostasis or in response to challenge and injury. For example, hematopoietic stem cells generate all mature blood cells, including monocytes, which have long been thought to be the major source of tissue macrophages. Recently, however, major macrophage populations were found to be derived from embryonic progenitors and to renew independently of hematopoietic stem cells. This process may not require progenitors, as mature macrophages can proliferate in response to specific stimuli indefinitely and without transformation or loss of functional differentiation. These findings suggest that macrophages are mature differentiated cells that may have a self-renewal potential similar to that of stem cells.

Faced with constant challenges from the environment and the physiological aging process, the body continuously replaces its cells. This occurs particularly for tissues that are most exposed to the environment, such as the mucosal epithelia, skin, and immune system. Mature differentiated cells from these tissues are continuously lost, and their life span can be very short: just a few days for blood monocytes, for example. Consequently, the turnover of these cells is enormous, even under conditions of normal tissue homeostasis but especially after tissue damage or in the case of the immune system, upon infection. Generally, this need for new cells is sustained by tissue-specific stem cells that are endowed with self-renewal capacity and by transient amplifying progenitor cells that further increase cell numbers during differentiation. Once cells reach full maturity, they typically cease proliferation. Thus, the constant replacement of mature cells normally depends on the input from self-renewing stem cells. One of the best-studied examples of such adult tissue-specific stem cells is hematopoietic stem cells (HSCs) that give rise to all blood cells and immunocytes.

Tissue macrophages serve important roles in the immune response, tissue homeostasis, metabolism, and repair. Because of these multifaceted activities, macrophages have been identified as key players (and targets) in diseases with major importance for public health, such as cancer and cardiovascular, autoimmune, chronic inflammatory, degenerative, and metabolic diseases (1). Like all other leukocytes, macrophages were thought to principally develop from hematopoietic stem cells via bone marrow progenitors and blood monocyte intermediates. This view has recently changed (Fig. 1) with the demonstration that major macrophage populations can be derived from embryonic progenitors in the yolk sac (2, 3) or after establishment of definitive hematopoiesis (4, 5) and can be maintained long-term, independently of input from adult hematopoietic stem cells (4, 6, 7). At the same time, it became clear that macrophages can proliferate and massively expand under challenge conditions, such as infections (7, 8), and that genetic inactivation of cell cycle withdrawal mechanisms enables indefinite self-renewal of mature macrophages without transformation or loss of macrophage function (9). Together, these recent data suggest that macrophages are mature differentiated cells that may be endowed with a self-renewal capacity akin to that of stem cells.

Multiple Pathways to Macrophages

Monocyte-Derived Macrophages

Before our recent understanding that tissue macrophages can be of embryonic origin, the idea that tissue macrophages originate from the bone marrow via circulating blood monocyte intermediates was textbook knowledge. Arguments to support this view had come mainly from bone marrow transplantation of experimental animals and humans, who received whole-body irradiation or other myelo-ablative treatments, such as cyclophosphamide chemotherapy. Radio-labeled bone marrow cells could be traced to skin implants and the peritoneal cavity of irradiated rats (10, 11). In mouse bone marrow chimeras, donor origin of tissue macrophage populations was demonstrated via the use of cytogenetic chromosome (12) or haplotype markers (13). In human patients receiving sex-mismatched bone marrow transplants, following the Y chromosome demonstrated the donor origin of alveolar macrophages (14).

Careful kinetic studies using radio-labeling of highly proliferative bone marrow progenitors and tracing the label to monocytes and macrophages also supported the bone marrow origin of macrophages (15). A radiolabeling approach during steroid-induced monocytopenia also provided evidence that monocytes give rise to pulmonary macrophages (16) and peritoneal macrophages after sterile inflammation (17). Based on this evidence, van Furth and Cohn proposed a model in which tissue macrophages arise from monocyte progenitors in the bone marrow via blood monocyte intermediates (15). These early observations were revisited more systematically in studies that demonstrated nearly complete reconstitution of several major tissue macrophage populations in irradiated bone marrow chimeras after 2 to 12 months, with the exception of microglia cells, which showed only a minor but detectable donor contribution at later time points (18, 19).

Adoptive transfer and tracing of labeled monocytes have also been employed to study monocyte-derived macrophages. Modern flow cytometry and green fluorescent protein (GFP)–reporter mice have made it possible to clearly characterize monocytes, of which two main subtypes exist: commonly named classical or inflammatory and nonclassical or patrolling monocytes, with a Ly6Chi, CCR2hi,CX3CR1lo or Ly6C,CCR2, CX3CR1hi surface marker phenotype, respectively (20), and a subtype-specific gene-expression profile (21, 22). Although monocytes have important functions inside blood vessels in their own right (21, 23, 24), these cells also leave the blood to infiltrate tissues and convert into macrophages under diverse challenge conditions such as atherosclerosis (25), myocardial infarction (26), muscle injury (27), or infection and inflammation (28). Upon irritation, aseptic wounding, or infection, a massive efflux of monocytes into neighboring tissue occurs within 1 to 2 hours or the first 24 hours, depending on the monocyte subtype (21). Consistent with historic carbon-particle–labeling experiments (29), modern-day in vivo labeling with fluorescent beads (30, 31) or transplantation of GFP-expressing monocytes (22, 3235) has demonstrated monocyte infiltration into inflamed skin, the peritoneal cavity, brain, and intestinal lamina propria (22, 30, 31, 34, 35) and noninflamed lung and intestines (22, 30, 32, 33).

Embryonic Progenitor–Derived Macrophages

Some data have long conflicted with the monocyte-centered view of macrophage origin. For example, the bone-seeking radio-element 89Sr, which incorporates close to bone marrow niches and selectively ablates HSC-dependent monopoiesis, did not reduce tissue macrophage populations in the lung, peritoneum (36), and liver (37). Similarly, CCR2 inactivation severely depletes circulating monocytes without substantially affecting tissue macrophage populations (7). Consistent with this observation, many tissue macrophage populations appear to be intact in patients with monocytopenia caused by leukemia or certain immune deficiency syndromes (3840). These observations, however, are not fully conclusive, as increased life span of downstream progeny can compensate for reduced monocyte input (41).

More direct evidence for sources of tissue macrophages, other than bone marrow–derived monocytes, comes from modern lineage-tracing experiments. In these experiments, Cre-recombinase is expressed under the control of a lineage-specific locus to excise a loxP-flanked Stop cassette from a fluorescent protein reporter. Consequently, all progeny of cells that expressed the marker in the past will be labeled, even after expression of the marker itself has ceased. A useful marker is CX3CR1, which is expressed in monocyte dendritic cell progenitors in the bone marrow, but not in many mature macrophage populations. Postnatal induction of Cre led to extensive labeling of monocytes but essentially no labeling of Langerhans cells; Kuppfer cells; or peritoneal, alveolar, or splenic macrophages—labeling would be expected if these populations were replaced by monocytes. By contrast, labeled macrophages could be found in the peritoneal cavity after thioglycollate-induced monocyte infiltration (41). Similarly, other lineage-tracing models with strong labeling of monocytes, such as S100a-, Flt3-, and Mx-Cre reporter mice, also showed only limited labeling in lung and spleen macrophages. This finding is supported by studies in parabiotic mice that revealed a poor contribution to lung, spleen, and peritoneal tissue macrophages from partner mouse monocytes (7). Together, these experiments indicate little or no contribution of monocytes to several tissue macrophage populations.

Parallel to these experiments, lineage-tracing experiments identified alternative developmental origins and suggested that many adult macrophage populations are derived from embryonic progenitors (2, 3, 41). Hematopoiesis in the embryo occurs in several waves, with primitive hematopoietic cells first arising in the yolk sac blood islands around embryonic day seven (E7), shifting later to the aorta-gonad-mesonephros region and the fetal liver before it is established in the bone marrow after birth. Yolk sac hematopoiesis produces only macrophages and erythroid cells. Pulse labeling with Runx-Cre ancestry mice at E7.5, before the development of HSCs and thus the establishment of definitive hematopoiesis, marked ~30% of adult brain microglia cells, similar to the proportion of marked yolk sac macrophages and microglia in the embryo (2). This demonstrated that brain microglia cells are derived from such early yolk sac macrophage progenitor cells.

Genetic approaches that selectively disable definitive hematopoiesis now indicate that beyond brain microglia, early yolk sac macrophage progenitors contribute more broadly to tissue macrophage populations in the adult (3). The transcription factor c-Myb is essential for HSC, but its inactivation does not affect yolk sac progenitors and primitive macrophage differentiation (3, 42). Despite the complete absence of definitive HSC-dependent hematopoiesis, tissue macrophage populations develop in multiple organs of Myb-deficient embryos and display a characteristic macrophage gene and F4/80hi surface-marker expression profile (3). Pulse labeling of cells expressing the macrophage colony-stimulating factor receptor (CSF-1R) at E8.5 in inducible Cre-ancestry mice confirmed that early embryonic progenitor–derived F4/80hi tissue macrophages can persist into adulthood (3). This is consistent with observations that in Myb mutant zebrafish postembryonic tissue, macrophage populations develop in the absence of definitive hematopoiesis (43). Together, these experiments show that early embryonic progenitor–derived macrophages can persist in tissues to adulthood. In addition to yolk sac progenitors, after the onset of definitive hematopoiesis a second wave of embryonic progenitors can contribute to epidermal Langerhans cells (4, 5), which have recently been classified as macrophages on the basis of their dependence on macrophage colony-stimulating factor (M-CSF, also known as CSF-1) (44). To what extent different populations of yolk sac progenitor–derived macrophages may subsequently be replaced by later embryonic progenitors or monocytes remains a matter of debate (45).

Context-Dependent Macrophage Origin

It is now established that macrophages can be of dual origin from embryonic progenitors or blood monocytes (Fig. 1) and that monocytes contribute only minimally to many tissue macrophage populations under homeostatic conditions (3, 7, 41, 46, 47). However, monocyte contribution can be strongly increased upon inflammation, and it remains an important question to what extent monocyte- and embryo-derived tissue macrophages are distinct and whether under physiological or pathological conditions conversion is possible.

It has been argued that monocytes maintain their own identity, even after entering the tissue, and do not convert into macrophages (7, 46, 48). Inflammatory monocyte–derived populations can be phenotypically and functionally distinguished from resident macrophages in many tissues (3, 7, 46, 4951) and, in some cases, disappear without contributing to the resident pool (49). On the other hand, lineage tracing revealed that thioglycollate-elicited macrophages derived from inflammatory monocytes changed phenotype to integrate into the resident macrophage pool and persisted over time (41). Gene profiling has also indicated that monocytes acquire macrophage gene expression after inflammation-induced tissue infiltration (21, 22, 46). Furthermore, adult Langerhans cells show no monocyte contribution in homeostatic settings (3, 52) but can show substantial replacement from the circulation during inflammation (31, 52), even under mild conditions (53). Monocytes can also fully replace resident macrophages, including F4/80hi populations that are normally embryo-derived, after myelo-ablative conditioning and bone marrow transplantation (7, 18, 19, 47, 52), demonstrating the capacity of monocytes for such an identity change. Monocytes can also contribute to macrophages under noninflamed conditions, but only in a highly tissue-specific manner (Fig. 1). Thus, in models involving no inflammation or irradiation injury, bone marrow transplantation results in a moderate to major donor contribution to F4/80hi tissue macrophages of the spleen, pancreas, lung, and kidney, but not other tissues (3). Lineage tracing and adoptive transfer also demonstrated a clear monocyte contribution to macrophages of the dermis (46) and intestinal lamina propria (22) but a lack of contribution to many other tissues (3, 7, 41, 46).

Fig. 1 New view of macrophage origin and self-renewal.

Macrophages (blue) can develop from early embryonic progenitors in the yolk sac (and, in some cases, after onset of fetal liver hematopoiesis) to be maintained throughout development and in adult tissues by local self-renewal (top). Similar to stem cells, local self-renewal of macrophages is characterized by low proliferation rates under steady-state conditions (small circular arrows) but strong expansion during development, after depletion, or under challenge (large circular arrows). After hematopoiesis shifts from the yolk sac to the fetal liver and the adult bone marrow, macrophages can also develop from HSCs through transient amplifying myeloid progenitors (large circular arrows) and nonproliferative blood monocytes (bottom). Monocyte-derived macrophages can give rise to resident macrophages under certain conditions, such as bone marrow transplantation after irradiation. More typically, monocyte contribution to resident macrophages is highly tissue-dependent and varies from no contribution for brain microglia and epidermal Langerhans cells to complete monocyte origin for intestinal lamina propria macrophages. Although not a definitive marker, resident macrophages are often F4/80hi, whereas recruited monocyte-derived macrophages are generally F4/80lo. Based on current knowledge, tissues are indicated in increasing order of monocyte contribution to resident macrophage populations (top to bottom). GMP, granulocyte-macrophage progenitor; MDP, macrophage-dendritic progenitor; LC, Langerhans cells; RP, red pulp; LP, lamina propria.

Whether the contribution of monocytes to macrophages increases over the life span of the organism, perhaps due to repeated episodes of low-level inflammation, is an interesting question. Nonetheless, it is clear that in most tissues the rate of conversion would be very low and that tissue macrophage populations, irrespective of their yolk sac, embryonic or adult origin, persist over the long term in tissues and self-maintain with no or minimal input from circulating monocytes. This finding indicates that tissue macrophages require mechanisms for local self-renewal to maintain their numbers in homeostasis and under challenge.

Local Macrophage Proliferation

In Homeostasis

Macrophage differentiation from proliferative progenitors involves cell cycle exit, but experimentally, proliferation arrest can be overcome without loss of the differentiated phenotype (9). Macrophages can also be maintained long-term in tissues with little input from HSC-derived progenitors (35, 7, 47, 52). This was initially shown for microglia cells and Langerhans cells, two populations that are separated from the circulation by the blood-brain barrier and the basal lamina, respectively, and may thus have more limited access to leukocyte influx. Microglia cells develop from early yolk sac progenitors (54) and enter the developing brain, where they undergo massive proliferative expansion throughout embryonic development (2). In the adult, they remain largely quiescent without input from bone marrow progenitors. Langerhans cells develop from embryonic progenitors before birth (35) and are also maintained independently from bone marrow contribution in the adult (52). Directly after birth, they undergo a burst of proliferative expansion and then maintain low levels of homeostatic proliferation (4). Thus, the acquisition and maintenance of the required cell numbers in these two examples appear to be achieved by the proliferation of differentiated macrophages rather than the influx of progenitors. Resident peritoneal macrophages exhibit similar proliferative expansion in newborn mice and maintenance in the adult (50). For alveolar macrophages, historical evidence of cell division came from observations of human biopsies (55) and colony-formation capacity of hamster or human alveolar macrophages (38, 56). Later, 3H-thymidine incorporation analysis led to calculations suggesting that the majority of alveolar macrophages are maintained by local proliferation under steady-state conditions (57). Consistent with this finding, a recent study confirmed a significant proportion of proliferating macrophages (10%) in the steady-state lung (7).

Importantly, proliferation not only sustains macrophage numbers under homeostatic conditions but also mediates their rebound after severe depletion. A strong expansion of resident local macrophages and a several-fold increase in proliferation have been observed for splenic red pulp, bone marrow, and alveolar macrophages using genetic diphtheria toxin and clodronate-depletion models (7). Reestablishment of normal macrophage numbers by local proliferation has also been observed in less artificial settings of macrophage depletion, such as influenza virus infection (7) and inflammation-induced loss of resident peritoneal macrophages (50). Physiological or experimental depletion of Langerhans cell and intestinal macrophages leads to small clonal cell clusters, indicating that cells undergo a few cell divisions before reaching homeostatic numbers (33, 53). Local macrophage expansion appears not to be reliant on a dedicated progenitor population, as during recovery from cytotoxic depletion, a macrophage that had previously divided had the same probability of entering the cell cycle as a cell that had not (7). This suggests that all macrophages are equally capable of proliferation and is consistent with the observation that genetically modified macrophages with indefinite self-renewal potential can be cloned and recloned with high efficiency and low variation (9). Together, these data indicate that proliferation and expansion of mature differentiated macrophage populations maintain adequate cell numbers during development and homeostasis.

Response to Challenge

The ability of mature macrophages to proliferate becomes particularly evident under stress and challenge conditions that require a large increase in macrophage numbers. Macrophage proliferation is observed in a variety of human pathologies including atherosclerosis, kidney disease, acetaminophen-induced liver failure, and chronic lung inflammation (25, 5860). Experimentally, injury in the central nervous system or neurodegenerative disease leads to massive microgliosis due to local proliferation of microglia cells (6, 49). Similarly, atopic dermatitis results in proliferative expansion of otherwise largely quiescent Langerhans cells (4). Macrophage proliferation also occurs under nonpathological conditions of stress or challenge. For example, pregnancy leads to a strong increase in uterine macrophage numbers that is largely mediated by local proliferation (61), and wound healing of skeletal muscle is associated with proliferation of previously recruited macrophages (27).

Monocytes are essential for the defense against a broad range of microbial pathogens (28), but their accumulation at an infection site is principally sustained by influx from the blood. Classical early inflammatory signals of microbial infection generally do not permit proliferation of recruited monocyte-derived macrophages, with the notable exception of Ly6B+ macrophages in zymosan-induced peritonitis (62). The drastic loss of resident macrophages occurring in the inflammatory process, however, is compensated by local proliferation at later time points (7, 50). Even during classical inflammatory episodes, macrophage numbers may thus be controlled by an early recruitment phase and later proliferative phase, as recently demonstrated for atherosclerotic lesions (25).

In stark contrast to the inflammatory pathways involved in control of microbial pathogens, the response to helminths involves an entirely different cellular network with a central role for the T helper cell 2 (TH2) cytokines interleukin-4 (IL-4) and IL-13 that signal through the IL-4 receptor α (IL-4Rα) (63). Macrophages activated via the IL-4Rα are found abundantly at sites of helminth infection and allergic inflammation and yet are typically considered to be anti-inflammatory, raising important questions about their relation to inflammatory monocytes (20). In a murine model of nematode infection that induces a potent TH2-type immune response, there is a slow accumulation of macrophages in the pleural cavity where the parasites reside. Using bone marrow chimeras in which the pleural cell population was protected from irradiation damage, it was demonstrated that the pleural resident macrophages expanded by local proliferation, with little contribution from bone marrow–derived cells (8). Proliferation and accumulation were dependent on high levels of IL-4 and/or IL-13 and appear restricted to the infection site (64). The action of IL-4 is not limited to the resident population but also stimulates proliferation of monocyte-derived macrophages (8). Thus, IL-4 and/or IL-13 induced by helminth infection drive the expansion of a noninflammatory resident population or drive proliferation of recruited cells and simultaneously convert them to a less inflammatory macrophage phenotype.

Mechanisms of Macrophage Self-Renewal

Cytokines Inducing Macrophage Proliferation

Several cytokines can drive macrophage proliferation (Fig. 2). The central importance of M-CSF in the maintenance of macrophage numbers is demonstrated by natural M-CSF mutations in mice (op/op) and rats (tl/tl) [reviewed in (65)]. All macrophages express the CSF-1R, but because it is also expressed by progenitors, it is difficult using these mice to distinguish differentiation defects from reduced proliferation of mature cells. However, with the use of blocking antibodies or reconstitution protocols, a specific role for M-CSF in resident macrophage proliferation has been identified in the peritoneal cavity in the steady state and inflammation (62); during recovery after experimental depletion in the spleen, peritoneal cavity, and lung (7); and in the growing myometrium during pregnancy (61). IL-34, an alternative ligand for CSF-1R produced by brain neurons and keratinocytes, stimulates self-renewal of microglia and Langerhans cells. However, inflammation-induced Langerhans cell repopulation still requires M-CSF (66, 67). CSF-1R signaling is also essential for unlimited self-renewal of MafB- and cMaf-deficient (Maf-DKO) macrophages (9).

Fig. 2 Cytokine regulation of macrophage self-renewal in different settings.

Monocytes are recruited to tissues under classical inflammatory conditions (1). In macroparasite-infected individuals, TH2 cells release large amounts of IL-4 or IL-13 (2) that lead to massive expansion of resident macrophages (3) and IL-4Rα activation, as well as proliferation of recruited inflammatory macrophages (4). M-CSF can also induce limited proliferation of subsets of recruited macrophages (5), which, under certain conditions and depending on the tissue, can integrate into the resident macrophage pool (6) (see Fig. 1). Self-renewal of resident macrophages (7) during developmental expansion, homeostasis, or insult recovery can be driven by M-CSF or IL-34 in the brain and epidermis and by GM-CSF in the lung.

Beyond the central role of CSF-1R ligands, granulocyte-macrophage colony-stimulating factor (GM-CSF)/CSF-2 is critical for lung macrophage homeostasis (68) and the ability to repopulate the lung after lethal irradiation (7). GM-CSF also induces proliferation of peritoneal macrophages in vivo (69) and can sustain the long-term culture of nontransformed fetal liver-derived macrophage lines (70). Thus, M-CSF, IL-34, and GM-CSF all participate in the process of self-renewal or repopulation, but their contributions are context- and tissue-specific. Furthermore, clear evidence exists for M-CSF–mediated macrophage proliferation in disease settings such as allograft rejection (71), suggesting roles beyond steady-state maintenance.

Recently, a potent proproliferative action of IL-4 on macrophages in vivo has also been documented (8). Injection of IL-4 or IL-13 into the peritoneal cavity of mice induces an almost exclusive expansion of tissue resident macrophages through local proliferation that is not limited to the injection site but drives resident macrophage expansion throughout the body, including the liver and spleen (8, 64). Critically, the IL-4Rα is not involved in the steady-state proliferation of tissue resident macrophages (7, 62, 64), and IL-4–mediated macrophage proliferation is independent of the CSF-1R (64). Indeed, IL-4 appears to allow macrophages to divide beyond homeostatic levels maintained by M-CSF (64). With repeated or sustained IL-4 delivery, it is even possible to cause death due to tissue macrophage hyperplasia (72), suggesting that endogenous controls of IL-4–driven proliferation fail in this artificial long-term and high-dose delivery system. IL-4 availability itself may therefore be an important limiting factor in vivo.

Mixed wild-type and IL-4Rα–deficient bone marrow chimeras have demonstrated that IL-4 acts directly on macrophages to drive proliferation (64). Surprisingly, however, IL-4 drives minimal macrophage proliferation in culture, suggesting that additional cofactors are required in vivo. Although direct injection of IL-4 into lymphocyte-deficient mice induces macrophage proliferation (8), CD4 helper T cells are required for macrophage expansion during helminth infection (73). This suggests that T cells are an important source of IL-4 in natural settings and that IL-4–driven proliferation may be restricted to adaptive TH2 environments. It will be critical to determine whether IL-4–mediated macrophage proliferation also occurs in noninfection settings, where there is a growing appreciation that the overarching function of macrophages activated via the IL-4Rα is to maintain tissue homeostasis (74).

Intracellular Signaling and Transcription Factors Controlling Macrophage Proliferation

Investigation of the signaling events downstream of the cytokine receptors has been the natural starting point to identify the intracellular mechanisms that mediate macrophage proliferation (Fig. 3). Although most of this work has been done in culture, it has been shown in vivo that AKT signaling is important for IL-4–induced proliferation (75). The IL-4Rα can also induce C/EBPβ, peroxisome proliferator–activated receptor γ interferon regulatory factor 4, and Stat6 activation (76), but whether the known roles of these transcription factors in the macrophage activation state extend to proliferation is unclear. Dedicated branches of cytokine signaling may exist for differentiation, survival, activation, and proliferation, as has been shown for the CSF-1R (77). Indeed, IL-4 signaling via Stat6 can inhibit M-CSF–stimulated macrophage proliferation (78). In a different context, Stat5 activation is sufficient to mediate extended macrophage proliferation by GM-CSF (70). M-CSF–induced proliferation involves the activation of the Myc, AP-1, and Ets transcription factors (7981), the latter two of which respectively activate c-myc and c-myb (82, 83), two proproliferative factors. MafB and c-Maf represent a core module of macrophage transcription factors (48) that are up-regulated during macrophage differentiation and can induce cell cycle arrest (84, 85). In myeloid progenitors, this is mediated by direct inhibition of Myb (84, 85), but because tissue macrophages can develop and proliferate in the absence of Myb (3, 43), this mechanism may not play a role in regulating the proliferation of mature macrophages. Because MafB can also directly bind and inhibit Ets-1 (86), it may rather take part in the inhibition of Ets activity on proproliferative genes including c-Myc (80, 81, 82), occurring during cell cycle withdrawal of differentiated macrophages (80). Consistent with this finding, c-Myc is up-regulated in M-CSF– and GM-CSF–induced proliferation (9, 70, 87) and is required for the extended self-renewal capacity of Maf-DKO macrophages (9). It will thus be interesting to determine whether MafB, c-Maf, or their targets are regulated during episodes of physiological or pathological macrophage proliferation in vivo.

Fig. 3 Intracellular signaling pathways of macrophage self-renewal.

M-CSF stimulation of CSF-1R signaling can activate Ets transcription factors via Ras and Erk signaling, which, in turn, can activate D-type cyclins and Myc. MafB and c-Maf repress Ets factors, as well as M-CSF–dependent Myc and KLF4 activation. The cross-antagonistic regulation of p53 and p21 by Myc and KLF4 may be important for balanced cell cycle activation that enables self-renewal without causing tumorigenic transformation. IL-4–induced phosphatidylinositol 3-kinase (PI3K) signaling is important for its proproliferative activity, and Stat5 can mediate GM-CSF–induced macrophage proliferation. Other signaling pathways induced by the cytokines but not documented as involved in proliferation are not pictured. Numbers indicate references on which the figure is based.

Do Macrophages Have Stem Cell–Like Self-Renewal Capacity?

The answer to this question hinges on the definition of self-renewal. Whereas in immunology this term is often relatively loosely employed as replacement of a certain cell population, in stem cell research it is a defining criterion, designating a cell division where at least one of the daughter cells has the same identity as the parental cell (88). Usually the term is thus reserved for stem cells, which can maintain self-renewal divisions over the long term, whereas highly proliferative progenitor cells change their identity as they differentiate. Local proliferation of tissue macrophages may qualify as self-renewal, not only in the loose (immunological) sense but also in the more strict (stem cell) sense, as cell cycle entry appears to occur independently of whether cells divided previously (7), suggesting that it is not restricted to a dedicated progenitor population. Consistent with this observation, Maf-DKO macrophages can divide indefinitely without loss of macrophage identity and can be cloned and recloned at the same efficiency (9), indicating that self-renewal in the strict sense, without loss of cellular identity, is possible in macrophages. Other examples in which mature differentiated cells can reenter the cell cycle are hepatocytes (89) and memory cells in the lymphoid system (90).

The notion of true macrophage self-renewal is further supported by the molecular mechanisms involved. Similar to embryonic stem (ES) cells (91, 92), self-renewal in Maf-DKO macrophages depends on Myc and Kruppel-like factor 4 (KLF4) (9). Both Myc and KLF4 are part of the original transcription factor cocktail that can induce reprogramming of somatic cells to pluripotent stem cells (93). However, these transcription factors are not required for the induction of pluripotency and have been suggested to mediate self-renewal (93). KLF4 does not induce proliferation in macrophages (9) but may mitigate the transforming activity of Myc (94) by opposing its effect on p21 and p53 (93, 95). The balanced activities of Myc and KLF4 may thus be required to enable macrophage self-renewal. Given the similarities in the role of Myc and KLF4 in ES cell and macrophage self-renewal, it will be interesting to determine whether similar signaling and transcription factor networks are employed in the two cell types.

Future Perspectives

We have reviewed the evidence that, irrespective of their origin, tissue macrophages can self-renew by local proliferation without replacement via hematopoietic stem cell–derived monocyte intermediates. It is important to appreciate how completely this concept breaks with the classical view of tissue maintenance by adult tissue-specific stem cells. The observations suggest that macrophages control their numbers independently of stem cells and in intimate relation with the tissue where they reside. It will be important to explore the relative contribution of local proliferation and monocyte recruitment in a range of inflammatory and noninflammatory settings and how the selective control of local macrophage proliferation versus monocyte recruitment could be turned to therapeutic benefit; for example, in excessive or chronic inflammatory conditions. To achieve this, we will need to understand the signaling pathways and cellular communications that control macrophage proliferation in vivo; in particular, the differences between a long-lived and largely quiescent homeostatic state and high rates of proliferation in response to challenge. It will be valuable to determine whether resident tissue macrophages employ similar signaling pathways and regulatory circuits as stem cells to respond to these divergent demands. It will also be important to understand how macrophage self-renewal differs from transient proliferation or tumorigenic transformation. If this succeeds, macrophages may teach us how to manage nontumorigenic cellular expansion ex vivo and in situ for tissue maintenance and regeneration.

References and Notes

  1. Acknowledgments: M.H.S. was supported by the Agence Nationale de la Recherche (ANR-11-BSV3-0026) and is a Fondation pour la Recherche Médicale (DEq. 20071210559 and DEq. 20110421320) and INSERM-Helmholtz group leader. J.A. received funding from the Medical Research Council UK (MR/K01207X1 and MR/J001929/1), the Wellcome Trust, and European Union FP7 Health-2009-4.3.1-1, 242131 (E PIAF).
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