INTRODUCTION
Macrophages are one of the most important players in innate immunity. However, new roles, unrelated to immunity, have been recently assigned to these particular cells. These roles can exhibit a wide range of phenotypes and functions depending on their environmental context.
Macrophages have been known for a long time to be associated with skeletal muscle regeneration (16,25) and several in vivo studies have shown that they actually participate in the muscle repair process. Indeed, by using several injury models (hindlimb ischemia, freeze-injury, unloading/reloading sequences, myotoxic agent injection), we and others have observed that the reduction of monocyte/macrophage entry into injured muscle hinders muscle regeneration, as shown by a delay in the appearance of regenerating myofibers and the persistence of intramuscular adipocytes (2,23,27,28). We have furthermore demonstrated by using transgenic diphtheria toxin receptor (CD11b-DTR) mice (in which monocytes/macrophages may be selectively depleted by diphtheria toxin injection) that total impairment of monocyte recruitment into damaged skeletal muscle during the first 24 h after injury totally prevents the muscle repair process, indicating the indispensable role of macrophages in muscle repair (2). These in vivo studies support the concept that inflammation triggered by macrophages after skeletal muscle damage is beneficial for muscle regeneration. We have performed several studies aimed at understanding the relationships that macrophages establish with myogenic cells, and more specifically, with myogenic precursor cells that are responsible for the regeneration of myofibers (10).
SKELETAL MUSCLE RESIDENT MACROPHAGES AND MONOCYTE RECRUITMENT AT TIME OF INJURY
In our first in vitro study, we demonstrated that myogenic cells are chemotactic for monocytes, acting through five identified molecular chemotactic systems: macrophage-derived chemokine, monocyte chemoattractant protein 1 (MCP-1), fractalkine (CX3CL1), vascular endothelial growth factor, and the urokinase system (6). Such chemotaxis may take place because activated myogenic cells are located in the vicinity of capillaries (7) and thus may chemoattract circulating monocytes. However, in vivo studies (M. Brigitte et al., unpublished data, 2008) have shown that the huge amounts of monocytes/macrophages that are recruited into muscle at the time of injury come from a specific muscle tissue compartment: the epimysium/perimysium (also called fascia). Analysis of this connective tissue has shown that the resident macrophages of skeletal muscle do reside in epimysium/perimysium and that they express MCP-1 (a chemoattractant for circulating inflammatory cells) upon muscle injury. Kinetics analysis of the monocyte/macrophage population showed that these cells accumulate early on in epimysium/perimysium before invading the muscle tissue itself. Moreover, we used chimera mice: CD11b-DTR mice were irradiated and engrafted with bone marrow from Tg:CAG GFP mice, in which all cells express green fluorescent protein (GFP) under the cytomegalovirus enhancer, chicken actin enhancer-promoter, and rabbit-globin poly(A) signal (CAG) promoter. In these chimera mice, only resident macrophages bear DTR, and not circulating GFP monocytes. We showed that selective depletion of these resident macrophages dramatically reduces monocyte/macrophage recruitment in the muscle compartment. These results show that, as in other tissues such as liver (11), resident macrophages are crucial for the subsequent recruitment of circulating monocyte cells to the injury site (Fig. 1).
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Figure 1: Monocyte/macrophage recruitment into skeletal muscle after toxic injury. Once activated upon muscle damage, myogenic cells secrete chemotactic factors for circulating monocytes (thin arrow). However, histological analyses have shown that the main route of monocyte/macrophage recruitment into muscle occurs via the epimysium and perimysium, in which resident macrophages are located (thick arrows). Upon muscle injury, resident macrophages secrete monocyte chemoattractant protein 1 (MCP-1) that allows the recruitment of both neighboring resident macrophages and circulating monocytes that further invade skeletal muscle. Macrophage-derived chemokine (MDC), MCP-1, fractalkine (CX3CL1), vascular endothelial growth factor (VEGF), and the urokinase system (uPA).
PHENOTYPE OF MACROPHAGES DURING MUSCLE REGENERATION: RESOLUTION OF INFLAMMATION
Macrophages are renowned for their heterogeneity, as reflected by the various specialized functions they adopt in different anatomical locations (9,26). Many of these activities seem to be opposing in nature (e.g., proinflammatory vs anti-inflammatory, immunogenic vs tolerogenic, and tissue destructive vs tissue repair activities) (26). Different activation states have been described for macrophages in vitro, each being associated with a particular phenotype and function, supposed to parallel various in vivo contexts, although evidence is lacking for many in vivo situations (9,15). Classical activation (also called M1) induces production of proinflammatory cytokines and reactive oxygen species and reflects the primary state of macrophage activation upon tissue injury or immune conflict. Alternative activation (also called M2a), observed in a Th2 environment, is associated with chronic inflammation, but evidence has been obtained mainly during parasitic infections. Anti-inflammatory or deactivation (also called M2c) is related to the phase of tissue repair during which macrophages secrete transforming growth factor-β (TGF-β). Various studies have established, both in vivo and in vitro, that macrophages have the ability to adapt to a changing microenvironment and therefore adopt new phenotypes (26).
We have shown that injured skeletal muscle recruits rapidly (in 1-2 days) mainly one population of circulating monocytes that become macrophages presenting an inflammatory profile. We have demonstrated that these macrophages switch their phenotype in situ in 1 to 3 days after their recruitment (Fig. 2). We have documented that this change occurs within the muscle tissue because the anti-inflammatory macrophages present in regenerating muscle 3 d after injury are not recruited from the blood but are instead derived from inflammatory macrophages. We have demonstrated in vitro that such a phenotype switch is obtained after phagocytosis of either apoptotic or necrotic myogenic cells. We therefore propose that in vivo, the switch from inflammatory to anti-inflammatory macrophages is likely caused by phagocytosis of both apoptotic and necrotic myofibers, in addition to other environmental cues that have been shown to trigger the switch from inflammation to healing by macrophages (Fig. 2) (21). It has been recently demonstrated in myocardium that two waves of monocyte subsets are subsequently recruited into the injured tissue (17). Nevertheless, this also leads to a switch of the macrophage phenotype present in the tissue from an inflammatory profile to an anti-inflammatory profile.
Figure 2: Switch of macrophage phenotype upon phagocytosis. Macrophages that invade skeletal muscle soon after injury present an inflammatory phenotype, characterized by high expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and secretory leukocyte protease inhibitor (SLPI), which are expressed by macrophages that have received proinflammatory cues.
In vitro, phagocytosis of both apoptotic and necrotic debris switches the phenotype of proinflammatory macrophages into anti-inflammatory macrophages, which express high levels of transforming growth factor-β (TGF-β), interleukin 10 (IL-10), and peroxisome proliferator-activated receptor-γ (PPAR-γ) that have been shown to be expressed by macrophages associated with resolution of inflammation and tissue repair (for details on these markers of inflammation, see Arnold
et al. 2).
These results show that postinjury muscle regeneration is characterized by sequential macrophage populations. First in the tissue are inflammatory macrophages, which are associated with monocyte recruitment and removal of necrotic material. Next, anti-inflammatory macrophages appear and are associated with healing and tissue repair.
MACROPHAGES AND SKELETAL MUSCLE REPAIR
We have shown that the specific depletion of intramuscular F4/80hi (a monocyte/macrophage marker whose expression increases with differentiation) macrophages during the phase of muscle repair (using CD11b-DTR mouse) results in a decrease in the diameter of regenerating myofibers. This result indicates that these anti-inflammatory macrophages are involved in muscle cell differentiation and/or fiber growth (2).
With the exception of regulation of inflammation, studies documenting a direct role of macrophages on modulating cell behavior are scant and include intestinal progenitor proliferation (19), erythroblast proliferation and maturation (20), and oligodendrocytic differentiation and myelination (8). In vitro studies have shown that the macrophage activation state may direct neural progenitor differentiation toward either neurogenesis or oligodendrogenesis (5).
We have performed a series of in vitro experiments using human myogenic cells and macrophages aimed at identifying the role of macrophages on myogenic cell behavior (2,6,24). We first identified that macrophages stimulate in a dose-dependent way the growth of myogenic cells. They are more efficient when the two cell types are allowed to contact each other. We showed that this stimulating effect is caused by: 1) the release of mitogenic factors for myogenic cells, which are likely growth factors known to be secreted by macrophages and to which myogenic cells respond efficiently (10); and 2) the establishment of cell-cell contacts that protect myogenic cells from apoptosis. We have identified four molecular systems involved in these cell contacts: vascular cell adhesion molecule 1-very late antigen 4, intercellular cell adhesion molecule-leukocyte function-associated molecule 1, platelet-endothelial cell adhesion molecule 1 (PECAM-1-PECAM-1), and CX3CL1-CX3CR1. These systems are more strongly expressed by myotubes (multinucleated differentiated myogenic cells) that are more protected from apoptosis compared with undifferentiated myoblasts. This suggests that macrophages could help protect these cells until they establish a final association with the matrix.
We further analyzed the effects of differentially activated macrophages. We used proinflammatory and anti-inflammatory activated human macrophages, the counterparts of inflammatory and anti-inflammatory cells observed in vivo during muscle regeneration in mouse, respectively (9). Our results substantiate the view that macrophage function may be related to activation state. In coculture experiments, inflammatory macrophages stimulate myogenic cell proliferation and inhibit their differentiation. Conversely, anti-inflammatory macrophages exhibit a strong differentiating influence on myogenic cells as assessed by both stimulation of the myogenic program and increased myoblast fusion (2). These results strongly suggest that the activation state of macrophages may modulate the myogenic process.
The in vitro process of myogenesis involves three main steps including cell differentiation per se (cell cycle withdrawal and expression of the myogenic program), migration of cells toward each other, and eventually fusion into multinucleated structures. We have analyzed the effects of macrophage-conditioned medium and showed that these three steps are differentially regulated by proinflammatory and anti-inflammatory macrophages (B. Chazaud, and H. Yacoub-Youssef, unpublished data, 2008). Medium from proinflammatory macrophages exerts negative effects on both myogenic differentiation and fusion while stimulating myogenic cell motility, which may be detrimental for cell fusion (3). Conversely, medium from anti-inflammatory macrophages stimulates both the myogenic differentiation and fusion processes (Fig. 3).
Figure 3: Effect of activated macrophages on in vitro myogenesis. Proinflammatory and anti-inflammatory macrophages differentially regulate in vitro myogenesis that includes three main steps: differentiation (i.e., expression of the myogenic program), migration of myogenic cells toward each other, and fusion itself. Upper panels: myogenic differentiation is inhibited by proinflammatory macrophages and stimulated by anti-inflammatory macrophages as shown by the expression of the myogenic transcription factor myogenin (left panel: arrows indicate the absence of myogenin expression in the nuclei of myogenic cells treated with proinflammatory macrophages; right panel: arrows indicate myogenin expression in the nuclei of myogenic cells treated with anti-inflammatory macrophages). Middle panels: myogenic cell mobility is strongly enhanced by proinflammatory macrophages compared with anti-inflammatory macrophages (each line on graph represents the route followed by an individual myogenic cell, reported relative to an x0/y0 origin). Lower panels: myogenic cell fusion is inhibited by proinflammatory macrophages and stimulated by anti-inflammatory macrophages as shown by the formation of multinucleated myotubes.
The molecular mechanisms involved in these processes are currently under investigation. It is likely that cytokines released by activated macrophages influence myogenic cell behavior. For example, tumor necrosis factor-α (TNF-α) is mitogenic for myoblasts and inhibits their differentiation (13). In vitro effects of TGF-β1 are more controversial, although in vivo neutralization of TGF-β1 in regenerating muscle was shown to reduce the diameter of regenerating myofibers (12). Beyond cytokines, cyclooxygenase 2 and its metabolites may also play a crucial role, as they have been shown to promote fusion of myoblasts and are necessary for good muscle repair (4,22).
CONCLUSIONS
Postinjury skeletal muscle regeneration is characterized by two distinct subsequent phases, each associated with different types of inflammatory cells. Soon after injury, infiltrating macrophages are classically activated and affect phagocytosis of tissue debris while preventing myogenic differentiation too early in the repair process. Then macrophages switch their phenotype to resolve inflammation, as has been shown in other tissues (21). The second phase of muscle repair is characterized by the presence of anti-inflammatory macrophages that while dampening environmental inflammatory signals, directly support myogenesis and myofiber growth. This tightly regulated sequence of macrophage phenotypes during muscle regeneration may explain the paradoxical effects of nonsteroidal anti-inflammatory drugs on the healing of muscle injuries. The drugs seem to be beneficial during the first days after injury while they induce impairment in functional capacity and histology when administered at later time points (18).
The word inflammation, used to describe an infiltrate of inflammatory cells into a tissue, covers a wide diversity of situations, and several recent studies have demonstrated that the inflammatory infiltrate may be beneficial for tissue repair (14,19,23,27). Our work describes the sequence that occurs in the context of the regeneration of a normal skeletal muscle after an injury induced by a myotoxic agent (notexin). Degenerative diseases of skeletal muscle are also characterized by the presence of infiltrates of inflammatory cells, including macrophages. It has been shown that macrophages participate in muscle degeneration during degenerative disease in the mouse (1). However, the phenotype and the role of macrophages remain to be fully elucidated in these pathological contexts.
Acknowledgment
This work was supported by INSERM, University Paris 12, Association Française contre les Myopathies, Fondation de France.
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