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Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems

Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion... Research Article 2497 Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems Corinne Sonnet*, Peggy Lafuste*, Ludovic Arnold, Madly Brigitte, Françoise Poron, François-Jérôme Authier, Fabrice Chrétien, Romain K. Gherardi and Bénédicte Chazaud INSERM E0011 ‘Cellular interactions in the neuromuscular system’, Faculté de Médecine, Institut National de la Santé et de la Recherche Médicale; Université Paris XII, 8 rue du Général Sarrail, 94000 Créteil, France *These authors contributed equally to this work Author for correspondence (e-mail: [email protected]) Accepted 22 March 2006 Journal of Cell Science 119, 2497-2507 Published by The Company of Biologists 2006 doi:10.1242/jcs.02988 Summary The mechanisms underlying stromal cell supportive survival pathways; (3) four pro-survival cell-cell adhesion functions are incompletely understood but probably molecular systems detected by DNA macroarray are implicate a mixture of cytokines, matrix components and expressed by macrophages and myogenic cells in vitro and cell adhesion molecules. Skeletal muscle uses recruited in vivo – VCAM-1–VLA-4, ICAM-1–LFA-1, PECAM- macrophages to support post-injury regeneration. We and 1–PECAM-1 and CX3CL1-CX3CR1; (4) macrophages others have previously shown that macrophages secrete deliver anti-apoptotic signals through all four adhesion mitogenic factors for myogenic cells. Here, we focused on systems, as assessed by functional analyses with blocking macrophage-elicited survival signals. We demonstrated antibodies; and (5) macrophages more strongly rescue that: (1) macrophage influx is temporally correlated with differentiated myotubes, which must achieve adhesion- the disappearance of TUNEL-positive apoptotic myogenic induced stabilisation of their structure to survive. cells during post-injury muscle regeneration in mice; (2) Macrophages could secure these cells until they establish direct cell-cell contacts between human macrophages and final association with the matrix. myogenic cells rescue myogenic cells from apoptosis, as assessed by decreased annexin V labelling and caspase-3 activity, and by increased DIOC-6 staining, Bcl-2 Key words: Skeletal muscle, Myogenic precursor cells, Macrophage, expression and phosphorylation of Akt and ERK1/2 Apoptosis, Cell adhesion molecules Introduction components and cell adhesion molecules (Charbord and Stromal cells are microenvironmental cells defined by their Moore, 2005). ability to support the development, maintenance, proliferation The spectrum of stromal cells is debated and remains ill and differentiation of tissue-specific cell types. For example, defined (Muller-Sieburg and Deryugina, 1995). Nevertheless, supportive stromal cells are crucially involved in macrophages (MPs) constitute one definite cell type in this haematopoiesis. The supportive stromal cell compartment is spectrum and were previously recognised to play a major role established before the formation of the haematopoietic system; in tissue repair and homeostasis maintenance (Gordon, 1995). it remains dynamic, instead of being static once established, In addition to their classical functions, including microbicidal reacting to extrinsic signals that can either damage or enhance activity, phagocytosis and antigen presentation, these stromal cell function and number (Muller-Sieburg and multifaceted cells efficiently support growth and differentiation Deryugina, 1995). In non-haematopoietic tissues, organogenic of other cell types (Gordon, 1995; Laskin and Laskin, 2001). processes are similarly driven by stromal cells that control Their supportive effect was documented with respect to parenchymal cell functions such as migration, proliferation, erythroblasts, hepatocytes, neurons, oligodendrocytes and differentiation and programmed cell death (Charbord et al., myogenic cells (Blasi et al., 1987; Cantini et al., 1994; 2000; Lapidot and Petit, 2002). Sadahira and Mori, 1999; Takeishi et al., 1999; Polazzi et al., The mechanisms underlying supportive functions of 2001; Gras et al., 2003; Chazaud et al., 2003b). stromal cells are incompletely understood. Stromal cells In contrast to bone marrow, where stromal cells are in place probably provide a complex molecular milieu that influences to support an ever-changing haematopoietic compartment, the behaviour of local stem cells, which have the choice skeletal muscle is normally a stable tissue that uses newly of many fates, including quiescence, self-renewal, recruited MPs to support post-injury muscle regeneration differentiation and apoptosis (Charbord and Moore, 2005). (McLennan, 1996; Pimorady-Esfahani et al., 1997; Lescaudron The molecules in this milieu are not well defined but probably et al., 1999). In a previous study, we found that, upon include a mixture of cytokines, extracellular matrix activation, a small myogenic stem cell population residing Journal of Cell Science 2498 Journal of Cell Science 119 (12) beneath the basal lamina of each adult myofibre, the so-called molecule 1 (ICAM-1; CD54) binding to leukocyte function muscle satellite cells (Mauro, 1961), can attract circulating associated molecule 1 (LFA-1); chemokine CX3CL1 binding monocytes and interplay with MPs to enhance their growth to CX3CR1; and platelet-endothelial cell adhesion molecule 1 (Chazaud et al., 2003b). In vitro studies suggested that MPs (PECAM-1; CD31) homophilic binding to another PECAM-1. can support myogenic precursor cell (mpc) growth by Finally, we used a mouse model of post-injury muscle stimulating their proliferation through soluble mitogenic regeneration to demonstrate spatiotemporal correlation factors, and by preventing their apoptosis through direct cell- between MP influx and fading of injury-induced mpc cell contacts involving unknown molecular systems (Chazaud apoptosis. et al., 2003b). MP-derived soluble factors inducing mpc proliferation have Results long been reported (Cantini et al., 1994; Cantini and Carraro, MPs inhibit both spontaneous and induced mpc 1995; Massimino et al., 1997), and the literature on myogenic apoptosis in a dose-dependent way cell growth factors is extensive (reviewed by Hawke and Garry, As assessed by annexin V labelling, the addition of human 2001). By contrast, the significance of direct contacts between monocyte-derived MPs to primary mpc cultures inhibited MPs and mpcs has not been previously explored in the setting spontaneous apoptosis of mononucleated myoblasts (Fig. 1A). of muscle regeneration. In fact, relatively little is known The protective effect of MPs was dose dependent (P<0.05), regarding the relevance of apoptosis to skeletal muscle apoptosis being inhibited by 75% at the 1:5 (mpc:MP) ratio homeostasis and repair, although evidence exists indicating (P<0.001) (Fig. 1A). Because the rate of spontaneous mpc that enhanced apoptosis plays a role during muscle aging, apoptosis was low (7.1±1.3% of the cells), further experiments muscular dystrophy, muscle denervation and unloading were performed after induction of mpc apoptosis by STS (reviewed by Jejurikar and Kuzon, 2003). Normal adult (Dominov et al., 1998; Columbaro et al., 2001). The protective myofibres are somewhat resistant to apoptosis. Their effect of MPs was strong enough to reduce STS-induced mpc sarcoplasm is refractory to mitochondrial cytochrome c- apoptosis (Fig. 1B). At the 1:10 (mpc:MP) ratio, apoptosis was dependent activation of type II caspases (Burgess et al., 1999). inhibited by 74% as assessed by annexin V labelling (P<0.001) Caspase-3 protein, which acts on the execution of cell death, (Fig. 1B) and by 60% as assessed by DIOC-6 staining is absent in normal myofibres (Ruest et al., 2002). Upstream (P<0.01). MPs inhibited STS-induced myoblast apoptosis in a protective mechanisms against apoptosis include blockage of dose-dependent and saturable way (P<0.001) (Fig. 1B). the two caspase-3 activation pathways, as the caspase-8 inhibitor ARC is expressed (Koseki et al., 1998), and caspase- The anti-apoptotic effect of MPs is more pronounced in 9 activator Apaf-1 is absent (Burgess et al., 1999), from myotubes skeletal muscle. The only physiological circumstance in which In culture, mpcs proliferate and give rise to mononucleated caspase-3 protein appears in adults is in regenerating muscle myoblasts that subsequently fuse with each other to form (Ruest et al., 2002). Such an expression of caspase-3 protein multinucleated myotubes. It is well established that myoblast in regenerating muscle is transient and might allow muscle to apoptosis occurs at times of serum deprivation used to boost get rid of excess replicating satellite cells or to delete myogenic differentiation. In addition, it has been shown that improperly innervated, newly formed myofibres (Ruest et al., myotubes are at particular risk of undergoing apoptosis upon 2002). In addition to its role in apoptosis, caspase-3 also stimulation with extrinsic stimuli as a result of their poor Bcl- participates to myofibrillar proteolysis (Du et al., 2004). Once 2 expression (Dominov et al., 1998; McArdle et al., 1999; regeneration is complete, caspase-3 mRNA remains detectable Ruest et al., 2002). Consistently, in our experiments, myotubes in the repaired muscle whereas caspase-3 protein becomes were 1.6-fold more sensitive to STS than myoblasts (P<0.05) undetectable (Ruest et al., 2002). Finally, from an evolutionary (Fig. 1C,D). Because the large size of myotubes precluded flow perspective, it seems important for skeletal muscle tissue to be cytometry analysis, determination of caspase-3 activity was protected from pro-apoptotic signals linked to exercise- used to compare the anti-apoptotic effect of MPs on STS- associated mitochondrial stress (Burgess et al., 1999) and, treated myoblasts and myotubes (Dominov et al., 1998). MPs consequently, mechanisms promoting restoration of the more efficiently rescued myotubes than myoblasts from STS- protected status of myogenic cells after muscle damage must induced apoptosis, as they decreased caspase-3 activity by 21% exist and could implicate stromal cells. in myoblasts and by 39% in myotubes [values at 5 hours, 1:2 We examined if and how MPs could play a significant role (mpc:MP) ratio, P<0.005] (Fig. 1C,D). in regulation of myogenic cell death during regeneration. We first extended our previous observations by analysing MP The anti-apoptotic effect of MPs is associated with protective effects against spontaneous and staurosporine activation of survival signalling (STS)-induced apoptosis of human mononucleated myoblasts Expression of the Bcl-2 anti-apoptotic protein is important for and multinucleated myotubes. Then, we selected candidate survival of expanding myogenic cells (Dominov et al., 1998). anti-apoptotic effector-counterligand molecular systems using As compared with mpc and MP cultures, co-cultures of mpcs DNA macroarray analysis, with confirmatory RT-PCR and with MPs showed enhanced expression of Bcl-2 (Fig. 2A). Pro- immunodetection in human MPs and mpcs. Four systems survival signalling pathways in myogenic cells include the previously implicated in cell-contact-mediated survival of mitogen-activated protein kinase and extracellular signal- other cell types were identified and shown to mediate in vitro regulated kinase (MAPK-ERK1/2) cascade, and the MP anti-apoptotic effects on mpcs by functional studies: phosphatidylinositol 3-kinase (PI 3-kinase) and serine/ vascular cell adhesion molecule 1 (VCAM-1; CD106) binding threonine protein kinase Akt/PKB pathway (Ostrovsky and to very late antigen 4 (VLA-4); intercellular cell adhesion Bengal, 2003; Reuveny et al., 2004). These pathways operate Journal of Cell Science Macrophages and myogenic cell survival 2499 Fig. 2. Induction of pro-survival signals in co-cultures Fig. 1. Inhibition of both spontaneous and induced mpc apoptosis by MPs. of mpcs with MPs. (A) Examples of immunoblots of (A,B) untreated (A) and STS-treated (B) mpcs were co-cultured with MPs at Bcl-2, phosphorylated Akt (Akt-P) and phosphorylated various ratios and mpc apoptosis was evaluated by annexin V labelling (white ERK1/2 (ERK1/2-P) in myoblasts (Mb), myotubes bars) and DIOC-6 staining (black bars) after exclusion of CD14 cells. In A, all (MT), MP cultures and co-cultures. Detection of -actin mpc:MP ratios used were statistically different from mpcs (1:0 ratio) (P0.04); was used to check the protein amount deposited in each in B, all mpc:MP ratios used, except the 1:0.5 ratio, were statistically different well. (B) Apoptosis of mpcs was detected with annexin from mpcs (1:0 ratio) (P0.07). (C,D) STS-treated myoblasts (C, Mb) and V (white bars) and DIOC-6 (black bars) in co-cultures myotubes (D, MT) were co-cultured with or without MP (1:2 mpc:MP ratio). of mpcs with MPs in the presence of H O at low 2 2 Apoptosis was evaluated by caspase-3 activity measurement. Results are means concentration (0.2 mM) or cytochalasin D (1 g/ml). ± s.e.m. of at least three experiments. Results are means ± s.e.m. of three experiments. through sequential phosphorylation events. Both pathways phosphokinase level determination [9.04±4.4 UI/ml in mpc were activated in co-cultures, as assessed by increased culture versus 7.3±3.8 UI/ml in co-cultures of mpcs with MPs phosphorylation of both ERK1/2 and Akt (Fig. 2A). (1:2)]. Altogether, these results substantiate the view that MPs To evaluate to what extent MP phagocytosis of damaged induce pro-survival signalling and decrease mpc apoptosis. cells (Geske et al., 2002) could have participated in the decreased number of apoptotic cells in co-cultures, we used DNA array in MPs and mpcs allows identification of four potent inhibitors of MP phagocytic activity, including H O at anti-apoptotic systems 2 2 low concentrations and cytochalasin D (Elliott and Winn, To select the cell-cell molecular systems at work in the 1986; Rubartelli et al., 1997; Anderson et al., 2002). The transduction of anti-apoptotic signals in mpcs, we used an addition of phagocytosis inhibitors to co-cultures did not mRNA profiling technique that allows analysis of a huge significantly modify the decreased rate of apoptotic mpcs number of genes at once. Among the 375 genes represented on observed in the presence of MPs (Fig. 2B). Consistently, the the DNA macroarray membrane we used, 12 (Table 1) had total number of mpcs did not significantly vary during the 6 products known to be involved in anti-apoptotic signals hour time of co-culture, as assessed both by cell count mediated by cell-cell contacts. [35,400±600 cells/cm in mpc culture versus 35,750±1300 Four of these products were constitutively expressed by cells/cm in co-cultures of mpcs with MPs (1:2)] and creatine human mpcs and had their counterligands expressed by human Journal of Cell Science 2500 Journal of Cell Science 119 (12) Table 1. Gene expression by human mpcs and MPs Intensity* GenBank mpcs stimulated by Expressed protein accession number mpcs MPs MPs versus mpcs Cadherin 5 (VE-cadherin) (homophilic) X79981 ND 304 PECAM-1 (CD31) (homophilic) M28526 106 602 1.5 ALCAM (CD166) (ligand) L38608 204 687 CD6 (receptor) U34625 ND 221 Fractalkine (CX3CL1) (ligand) U91835 90 89 CX3CR1 (receptor) U20350 57 14 1.2 VCAM-1 (CD106) (ligand) X53051 160 162 VLA-44 (CD49d) (receptor subunit) X16983 74 355 1.8 VLA-41 (CD29) (receptor subunit) X07979 464 929 1.2 ICAM-1 (CD54) (ligand) J03132 160 162 LFA-1L (CD11a) (receptor subunit) Y00796 74 355 2.2 LFA-12 (CD18) (receptor subunit) M15395 464 929 1.6 ND: not detected. *Arbitrary units. Intensity detected in mpcs stimulated by MPs (24 hours incubation in MP-conditioned medium as described in Chazaud et al., 2003b) versus intensity detected in mpcs. MPs (Table 1), as follows: (1) VCAM-1 binding to VLA-4 except PECAM-1, which could not be visualised in mpcs (41 integrin); this adhesion system mediates the protective despite positive detection by immunoblotting in differentiated effects of MPs to erythroblasts (Hanspal and Hanspal, 1994; mpcs (Fig. 3B). Expression of all four receptors was much Sadahira and Mori, 1999), of stromal cells to haematopoietic stronger in myotubes than in myoblasts (Fig. 3B). stem cells, B cells and plasma cells (Koopman et al., 1994; Oostendorp et al., 1995; Wang et al., 1998; Hayashida et al., CX3CL1-CX3CR1, VCAM-1–VLA-4, ICAM-1–LFA-1 and 2000; Minges Wols et al., 2002; Hall et al., 2004) and of PECAM-1–PECAM-1 are functional at the mpc surface endothelial cells to mast cells (Mierke et al., 2000). It is also Adhesion assays were used to assess functional availability of involved in protection of T cells and retinal ganglion cells the candidate molecular systems at the MP-mpc interface. It (Rose et al., 2000; Leussink et al., 2002; Leu et al., 2004). (2) was found that mpcs adhered on a MP monolayer in a dose- ICAM-1 binding to LFA-1 (L2 integrin); this system dependent, time-dependent and saturable fashion (Fig. 4A,B), mediates protective effects of endothelial cells to allowing the involvement of specific receptor-ligand transmigrating lymphocytes (Borthwick et al., 2003). Its interactions to be assessed. Blocking antibodies against both implication in support of bone marrow stromal cells to T cells CX3CL1-CX3CR1, VCAM-1–VLA-4, ICAM-1–LFA-1 or (Winter et al., 2001), and of follicular dendritic cells to B cells PECAM-1 failed to inhibit mpc adhesion on MPs significantly (Koopman et al., 1994) has been also reported, but in vitro (data not shown), suggesting robust redundancy of cell experiments have yielded somewhat discrepant results (Zen et adhesion systems involved in mpc-MP contacts. Therefore, al., 1996; Wang and Lenardo, 1997). (3) CX3CL1 (fractalkine) adhesion assays using mpcs deposited on coats of immobilised binding to CX3CR1; CX3CR1 uniquely binds membrane- ligands were used to test the functionality of each receptor. It anchored and shed soluble forms of CX3CL1 (Bazan et al., was shown that mpcs adhered in a dose-dependent and 1997), which are respectively involved in firm cell-to-cell saturable way to VCAM-1 (Fig. 4C), CX3CL1 (Fig. 4D), adhesion (Fong et al., 1998; Umehara et al., 2001) and in ICAM-1 (Fig. 4E) and PECAM-1 (Fig. 4F), as previously chemotaxis (Chapman et al., 2000). MPs and neural cells shown for other cell types (Meyer et al., 1995; Imai et al., 1997; reciprocally signal through this system to suppress apoptotic Bird et al., 1999; Goda et al., 2000). These results strongly cell death (Harrison et al., 1998; Boehme et al., 2000; Meucci suggested that VLA-4, CX3CR1, LFA-1 and PECAM-1 et al., 2000; Mizuno et al., 2003; Deiva et al., 2004). This expressed at the mpc surface were used to bind VCAM-1, system also prevents apoptosis in intestinal epithelium (Brand membrane-bound CX3CL1, ICAM-1 and PECAM-1, et al., 2002). (4) Homophilic PECAM-1 interactions; respectively. endothelial cell PECAM-1 prevents apoptosis of both neighbouring endothelial cells (Bird et al., 1999; Evans et al., CX3CL1-CX3CR1, VCAM-1–VLA-4, ICAM-1–LFA-1 and 2001; Gao et al., 2003) and transmigrating leucocytes (Ferrero PECAM-1–PECAM-1 mediate MP anti-apoptotic activity et al., 2003) through homophilic PECAM-1 interactions. on mpcs Exposure to MP-conditioned medium reinforced mRNA Co-cultures of mpcs with MPs were incubated with blocking expression of all counterreceptors by mpcs (Table 1). Results antibodies to assess anti-apoptotic effects of the selected of DNA macroarray were confirmed by RT-PCR. VCAM-1, molecules in our model. Blockage of each molecular system ICAM-1, CX3CL1 and PECAM-1 mRNAs were detected in inhibited the beneficial effects of MPs on mpc survival MPs (Fig. 3A). It was shown that mpcs, which are already (P<0.05) (Fig. 5), as assessed by increased annexin V labelling known to express the counterreceptor 1 integrin (Vachon et and caspase-3 activity, and decreased DIOC-6 staining al., 1997), expressed 4, L and 2 integrins, and CX3CR1 following exposure to blocking antibodies. Consistently, and PECAM-1 mRNAs (Fig. 3A). The corresponding proteins caspase-3 activity was increased in myotubes in the presence were immunodetected at the cell surface of MPs and mpcs, of the different blocking antibodies (Fig. 5C). Journal of Cell Science Macrophages and myogenic cell survival 2501 Influx of MPs and fading of mpc apoptosis are (Lefaucheur and Sebille, 1995). At 3 hours post-injury, synchronous during post-injury muscle regeneration TUNEL-positive nuclei were abundant in damaged areas (Fig. To examine the relevance of in vitro results, we injected snake 6A) (15.6 apoptotic cells/field). It was found that 60% (9.5 venom notexin in tibialis anterior muscle of adult mice cells/field) of TUNEL-positive cells were desmin negative and presumably corresponded to non-muscle cells; 40% (6.1 cells/field) of apoptotic cells were desmin positive and probably corresponded to activated satellite cells, interstitial myoblasts or myotubes (Fig. 6A). As MP influx proceeded in the regenerating muscle, the number of both the overall apoptotic cell population and of TUNEL-desmin double- positive cells dramatically decreased (Fig. 6A). At 48 hours post-injection, MP infiltration was massive and apoptotic mpcs were extremely rare, accounting for no more than 20% (0.12 cells/field) of the remaining apoptotic cells (0.62 cells/field) at this time (Fig. 6A). At this time point, both anti-apoptotic effectors VCAM-1, ICAM-1, CX3CL1 and PECAM-1, and their counterreceptors VLA-4, LFA-1, CX3CR1 and PECAM- 1, were expressed by MPs and mpcs, respectively, in the regenerating areas (Fig. 6B). Fig. 3. Expression of candidate effectors by human MPs and mpcs. (A) RT-PCR analysis of CX3CL1, VCAM-1, ICAM-1 and PECAM- 1 mRNA in MPs, and of CX3CR1, 4, L, 2 integrins, and PECAM-1 mRNA in mpcs. 2M is beta2microglobulin. Fig. 4. Functionality of candidate effectors at the mpc cell (B) Immunolabelling of CX3CL1, VCAM-1, ICAM-1 and membrane; adhesion assays. (A,B) Adhesion of mpcs on a MP PECAM-1 on MPs (left panel) and of CX3CR1, VLA-4 and LFA-1 monolayer according to incubation time (A) and mpc concentration on mpcs (right panel), revealed by DAB substrate kit for peroxidase. (B). Adhesion of mpcs on VCAM-1 (C), CX3CL1 (D), ICAM-1 (E) Magnification, 300. PECAM-1 expression in mpcs was assessed by and PECAM-1 (F) coats. Results are means ± s.e.m. of three immunoblotting. MT, myotube; Mb, myoblast. experiments. Journal of Cell Science 2502 Journal of Cell Science 119 (12) Discussion Muscle damage is known to induce massive MP infiltration In the present study, we demonstrate that: (1) MP influx is of the injury site (McLennan, 1996; Pimorady-Esfahani et al., temporally correlated with fading of mpc apoptosis during in 1997). Initially, the role of these blood-borne cells was believed vivo post-injury muscle regeneration; (2) MPs rescue to be limited to clearance of necrotic fibres (McLennan, 1996; differentiating mpcs more than cycling mpcs from apoptotic Pimorady-Esfahani et al., 1997). However, several in vivo and cell death; (3) MPs deliver anti-apoptotic signals to mpcs in vitro studies have shown that MPs are essential in through direct cell-cell contacts involving VCAM-1–VLA-4, orchestration of the muscle repair process (Grounds, 1987; ICAM-1–LFA-1, PECAM-1–PECAM-1 and CX3CL1- Lescaudron et al., 1999). CX3CR1 interactions. The decision of a cell to proliferate, differentiate or undergo apoptosis is an integrated response to its growth factors and adhesive environment (Schwartz and Ingber, 1994). At the population level, mpc growth depends on both cell-cycling activity and cell survival. Previous studies on mpc-supporting cues have focused on MP-released soluble growth factors (Robertson et al., 1993; Cantini and Carraro, 1995; Merly et al., 1999). Some particular growth factors, such as insulin growth factor I (IGF-I), in addition to being a potent myogenic differentiation factor (Tureckova et al., 2001), can both stimulate mpc proliferation in the presence of other soluble factors (Napier et al., 1999) and promote mpc survival (Lawlor and Rotwein, 2000). However, our previous (Chazaud et al., 2003b) and present studies indicate that MP-derived soluble factors globally stimulate mpc proliferation, as assessed by thymidine incorporation, whereas direct MP cell contacts confer protection against apoptosis to mpcs. Apoptotic cell death is a normal developmental event involving both proliferating myoblasts and postmitotic myofibres (Garcia-Martinez et al., 1993; Tidball et al., 1995; McClearn et al., 1995). As shown in our in vivo study, apoptosis of myogenic cells also occurs during regeneration of postnatal muscle. In this setting, TUNEL-positive myogenic cells disappear as MP infiltration proceeds. Obviously, this might reflect both MP phagocytosis of dead cells and the delivery of an MP pro-survival signal to living mpcs. In vitro, the protective effects of MPs were twofold stronger towards post-mitotic differentiating mpcs than towards cycling myoblasts. The physiological significance of this finding remains elusive. Furthermore, mpcs are at risk of undergoing apoptosis for different reasons during the proliferation and differentiation process. Fast-cycling myoblasts must face difficulties in maintaining adequate DNA repair that might constitute an intrinsic signal for apoptosis (Wang and Walsh, 1996). Then, as they withdraw from the cell cycle and begin to differentiate, mpcs are at particular risk of myoblast-fusion-associated apoptosis, induced by endoplasmic reticulum stress (Nakanishi et al., 2005). Finally, myotubes elongate through additional myoblast fusion and must progressively stabilise their structure by establishing close association with the extracellular matrix (ECM) (Huppertz et al., 2001). Myogenic cell adhesion to the Fig. 5. Functionality of candidate effectors at the mpc cell microenvironment seems to be crucial for their survival, as membrane; apoptosis assays. STS-treated mpcs were co-cultured demonstrated by increased muscle cell apoptosis associated with or without MPs in the presence or absence of antibodies with deficiencies in ECM-binding proteins such as 5 and directed against CX3CL1 and CX3CR1, VCAM-1 and VLA-4, 71 integrins, and ECM proteins such as laminins (Vachon ICAM-1 and LFA-1, or PECAM-1 (see bottom of figure). et al., 1996; Vachon et al., 1997; Miyagoe et al., 1997; Taverna (A,B) Apoptosis of mpcs was evaluated by annexin V labelling (A) et al., 1998; Montanaro et al., 1999). It is possible that MP- and DIOC-6 staining (B) after exclusion of CD14 cells. supportive cues help myotubes to achieve their adhesion- (C) Myoblast (white bars) and myotube (black bars) apoptosis was induced stabilisation safely. In line with this view, myotubes, evaluated by caspase-3 activity measurement. Results are expressed which poorly express Bcl-2 and are therefore more sensitive as percentage of apoptosis in STS-treated mpcs and are means ± s.e.m. of at least three experiments. than myoblasts to STS-induced apoptosis (Dominov et al., Journal of Cell Science Macrophages and myogenic cell survival 2503 Fig. 6. Apoptosis and muscle regeneration. Notexin-treated mouse muscle was labelled with a set of antibodies at different times after injury. (A) Example of apoptotic (red) and desmin + myogenic cells (green) at 3, 6 and 24 hours post-injury. MP infiltration was evaluated after F4/80 immunolabelling (blue curve) or CD11b immunolabelling (red curve) according to a 0-5 scale and the total number of apoptotic cells per field (black curve) was estimated. Among total apoptotic cells (white bars), apoptotic desmin-positive myogenic cells (black bars) was estimated at each time point. (B) Examples of immunolabellings of myogenic (desmin ) and macrophagic (CD11b ) cells for the anti- apoptotic molecular systems 3 days after injury. Blue: DAPI nuclei staining. Bars, 10 m. 1998), are endowed with stronger expression of the four MPs with mpcs showed increased Akt and ERK1/2 receptors involved in adhesion-induced pro-survival phosphorylation, increased Bcl-2 expression and decreased signalling. caspase-3 activity. VLA-4, but not LFA-1, PECAM-1 and CX3CR1, was We previously showed that, early after activation, mpcs previously reported to be expressed by mpcs and to increase secrete a set of chemoattractants to initiate recruitment of with myogenic differentiation (Rosen et al., 1992). The authors circulating monocytes into damaged muscle (Chazaud et al., evaluated VLA-4 as an ECM receptor (Rosen et al., 1992), 2003b). Once recruited, monocytes differentiate into MPs, although this integrin, like LFA-1, is also involved in cell-cell which are monocyte-derived MPs expressing VCAM-1, adhesion and signalling. All four receptors expressed by mpcs, ICAM-1, PECAM-1 and CX3CL1, as shown herein. The upon binding of their respective ligands VCAM-1, ICAM-1, newly recruited MPs release soluble factors that both amplify PECAM-1 and CX3CL1, were previously shown to mediate recruitment of MPs and stimulate mpc proliferation (Chazaud anti-apoptotic signalling in a variety of non-muscle cell types et al., 2003b). In addition, according to our DNA array, soluble (see Results section). VLA-4, PECAM-1 and CX3CR1 factors produced by MPs reinforce mpc expression of VLA-4, mediate activation of the PI 3-kinase/Akt survival pathway LFA-1, PECAM-1 and CX3CR1 by 20-80%. Thus, when MPs (Meucci et al., 2000; Gao et al., 2003; Ferrero et al., 2003; enter into contact with mpcs, both cell types appropriately Deiva et al., 2004) and, in addition, CX3CR1 activates ERK1/2 express anti-apoptotic ligands and counterreceptors. (Brand et al., 2002; Deiva et al., 2004). These signalling In conclusion, the present study highlights the complex pathways are both involved in mpc survival (Lawlor and network of intercellular signalling and communication Rotwein, 2000). Among many pro-survival effects, Akt involved in the organisation of the stromal support of phosphorylates BAD, causing its release from the complex it myogenesis. Our data indicating that inflammatory cells, i.e. forms with Bcl-2, allowing Bcl-2 to exert its anti-apoptotic macrophages, are beneficial for muscle regeneration are in activity freely (Song et al., 2005). Both Akt and ERK1/2 accordance with in vivo studies showing that blocking pathways lead to inhibition of caspase-3 (Allan et al., 2003; inflammation with anti-inflammatory drugs might be Song et al., 2005), the major effector of the last step of muscle deleterious for muscle regeneration and repair (Mishra et al., cell apoptosis (Tews, 2002). Consistently, co-cultures of 1995; Shen et al., 2005). Moreover, evidence that a set of Journal of Cell Science 2504 Journal of Cell Science 119 (12) adhesion molecules rescue mpcs from apoptosis might open the possibility of improving myoblast transfer therapy. A strong limitation of this therapeutic approach consists of early massive cell death of non-mechanical origin (Chazaud et al., 2003a), affecting >95% of transplanted mpcs (Skuk and Tremblay, 2000). Moreover, mpcs induced to proliferate actively ex vivo to obtain a huge number of cells for transplantation was shown to increase their susceptibility to undergo apoptosis upon deprivation of extrinsic supportive cues (Rehfeldt et al., 2004). It seems likely that the use of anti- apoptotic cells or molecules could limit massive transplanted cell death, thus allowing appropriated mpc proliferation, differentiation and striated muscle repair. Materials and Methods Cell cultures Unless indicated, culture media components were from Invitrogen (Gibco) and culture plastics from TPP AG (Trasadingen). Human mpcs were cultured from muscle samples as previously described (Chazaud et al., 2003b). Only cultures presenting over 95% CD56 cells (immunolabelling using anti-CD56 antibodies, diluted 1/20; Sanbio/Monosan) were used. Growing medium [HAM-F12 medium containing 15% fetal calf serum (FCS)] was used for culturing mpcs. To obtain myotubes, medium was replaced by HAM-F12 medium containing 5% FCS (differentiating medium) at time of subconfluence and cells were further cultured during 10 days (Lafuste et al., 2005). MPs were obtained from monocytes isolated from human blood as previously described (Chazaud et al., 2003b). Briefly, monocytes were seeded at 0.510 cell/ml in Teflon bags (AFC) in RPMI medium containing 15% human AB serum for 8 days. Cell treatments and co-cultures In each series of experiments, the number of mpcs remained constant whereas the number of MPs varied. Undifferentiated mpcs were seeded at 10,000 cells/cm . Differentiated myotubes were counted in order to seed the appropriate number of MPs, from 1:10 ratio. Co-cultures were incubated in growing or differentiating medium for 6 or 24 hours at 37°C. In some experiments, mpc apoptosis was first induced by staurosporine (STS) treatment (1 M for 6 hours). In some experiments, blocking antibodies were added in co-cultures of mpcs with MPs at saturating concentrations (calculated from IC50 or from previous studies): anti-CX3CL1 (3 g/ml, 51637.1 clone; R&D Systems) (Chazaud et al., 2003b), anti-CX3CR1 (15 g/ml, TP502; Torrey Pines Biolabs) (Chapman et al., 2000), anti-VCAM-1 (5 g/ml, 1G11 clone; Immunotech) (Minges Wols et al., 2002), anti-VLA-4 (5 g/ml, HP2/1 clone; Immunotech) (Hayashida et al., 2000), anti-LFA-1 (5 g/ml, TS1/22 clone; Endogen) (Hayashida et al., 2000), anti-ICAM-1 (5 g/ml, 84H10 clone; Fig. 7. Measurement of mpc apoptosis. (A) Example of flow Immunotech) (Winter et al., 2001), anti-PECAM-1 (5 g/ml, VM64 clone, cytometric analysis of mpc apoptosis in co-cultures of mpcs with Biodesign International). In other experiments, co-cultures were performed in the MPs. CD14 labelling is used to discriminate MPs from mpcs. The presence of hydrogen peroxide (0.2 mM; Sigma) (Anderson et al., 2002) or + – apoptotic mpc population is gated in red: annexin V CD14 cells cytochalasin D (1 g/ml, Sigma) (Elliott and Winn, 1986; Rubartelli et al., 1997). – – Controls included addition of whole IgGs from mouse and rabbit (3 g/ml; Vector and DIOC-6 CD14 cells. (B) Example of spontaneous (dotted Laboratories). lines) and STS-induced (continuous lines) apoptosis in mpc cultures. (C) Expression of CD14 by CD45 cells in co-cultures of mpcs with Measurement of mpc apoptosis by flow cytometry MPs. Trypsin was used to detach mpcs and detection of apoptotic cells was performed using annexin V plus CD14 labelling and DIOC-6 plus CD14 staining. CD14 labelling was used to exclude MPs detached by the trypsinisation procedure (Fig. 7A). Cells were resuspended in 100 l buffer (140 mM NaCl, 2.5 mM CaCl , 10 recovered after centrifugation at 4000 g for 10 minutes at 4°C. Protein concentration was determined using the BCA protein assay kit from Pierce. Aliquots mM HEPES pH 7.4) containing either 2 l of annexin V (Roche Diagnostics) or 70 nM of DIOC-6 (Molecular Probes) and 10 l of TRITC-conjugated anti-CD14 corresponding to 30 g of proteins were diluted in caspase-3 reaction buffer (1 M Hepes pH 7.4, 40 mM EDTA pH 8.0, 100 mM DTT, 25% sucrose) and incubated antibodies (RMO52; Immunotech) for 30 minutes. Cells were washed before analysis by flow cytometry on a FACSCalibur (BD Biosciences). Apoptosis of mpcs during 8 hours at 37°C in a microplate with caspase-3 substrate (Ac-DEVD-AFC fluorogenic substrate, Biomol Research Laboratories). Enzymatic activity was was significantly increased by STS treatment, reaching 30±13% of the cells (annexin V detection) and 44±13% of the cells (DIOC-6 detection) (P<0.01) (Fig. measured every 30 minutes with a fluorescence plate reader FL600 (Bio-Tek) and was expressed in arbitrary units. 7B). As the range of apoptotic mpcs was 19-60% (annexin V detection) and 30- 70% (DIOC-6 detection) of the cells, mpc apoptosis was expressed in percentage of apoptosis evaluated in STS-treated mpc cultures (without MPs). In co-cultures Adhesion of mpcs on MPs of MPs with untreated mpcs, CD14 expression was not affected (Fig. 7C); in co- Before being allowed to adhere on a confluent monolayer of MPs at various densities cultures of MPs with STS-treated mpcs, we observed no more than 4-5% of CD14 (5000 to 50,000 mpcs per well) and for various times (30 to 120 minutes), mpcs cells among CD45 cells (Fig. 7C), indicating that gating allowed exclusion of were labelled with 5-bromo-2-deoxyuridine (BrdU) for 72 hours. BrdU was then >95% of MPs. quantified using a colorimetric assay (Cell proliferation ELISA BrdU kit; R&D Systems). Measurement of caspase-3 activity Proteins from mpcs, MP cultures and mpc-MP co-cultures were extracted in lysis Adhesion of mpcs on ligand coats buffer (50 mM Hepes pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 40 mM EGTA pH Flat-bottomed 96-well sterile plates were coated with human recombinant VCAM- 8.0, 100 mM DTT, 2 g/ml leupeptin, 2 g/ml aprotinin, 1 g/ml pepstatin) and 1, CX3CL1, ICAM-1 or PECAM-1 (0.001 to 100 nM) (R&D Systems) in Journal of Cell Science Macrophages and myogenic cell survival 2505 phosphate-buffered saline (PBS). Non-specific binding sites were blocked with 1% In vivo immunolabellings bovine serum albumin for 30 minutes at room temperature. 30,000 mpcs per well Muscle cryosections were double labelled with either desmin antibodies (60 g/ml; were allowed to adhere for 2 hours at 37°C. Non-adherent cells were removed by Abcam) and anti-VLA-4 (15 g/ml, Chemicon International) or anti-LFA-1 (10 gentle PBS washes. Cells were fixed with acetone and methanol for 15 minutes and g/ml; Abcam) or anti-PECAM-1 (10 g/ml, Santa Cruz) or anti-CX3CR1 (10 stained with 0.5% Violet Crystal for 15 minutes. The number of adherent cells was g/ml; R&D Systems, using the MOM kit from Vector Laboratories) antibodies to evaluated by reading the OD at 540 nm. detect mpc expression. Slides were treated with anti-CD11b antibodies (10 g/ml; BD Biosciences) and anti-CX3CL1 (15 g/ml; Abcam) or anti-VCAM-1 (15 g/ml; R&D Systems) or anti-ICAM-1 (50 g/ml; Chemicon International) or anti- DNA array PECAM-1 (10 g/ml; Santa Cruz) antibodies to detect MP expression. To evaluate Total RNA was prepared from human mpcs and MPs using the RNeasy mini kit MP infiltration after injury, slides were treated with anti-CD11b as above or anti- (Qiagen). All further steps were performed according to the manufacturer’s F4/80 antibodies (20 g/ml; Abcam). Primary antibodies were detected with either instructions in the human cytokine array GA001 kit (R&D Systems). For mpc and cy3-labelled or FITC-labelled secondary antibodies (Jackson ImmunoResearch MP samples, 5 and 7 g of total RNA gave labelled cDNA of 600,000 and 800,000 Laboratories). Controls included incubation with whole IgGs from species of the cpm, respectively, which was deposited on membranes. Results were read using a primary antibody (Vector Laboratories). Slides were examined as described above. Phosphorimager (Amersham) after 72 hours exposure time. Analysis was performed using Image Quant software (Amersham), which allows background noise subtraction, correction for the variation of density for housekeeping genes and, Statistical analyses finally, for comparison of densitometric signals. Results were expressed in arbitrary Except DNA array, all experiments were performed using at least three different units. cultures or animals. The Student’s t-test and ANOVA analysis were used for statistical analyses. P<0.05 was considered significant. RT-PCR Total mpc or MP RNA (1.5 g) was reverse transcribed and amplified using This work was supported by the Association Française contre les OneStep RTPCR (Qiagen) and specific primers. For CX3CL1 [primers described in Myopathies and Fondation de France. We thank S. Lotersztajn for Lucas et al. (Lucas et al., 2001)], amplification was performed at 94, 64 and 72°C helpful discussions. for 30 seconds, 30 seconds and 1 minute, respectively, for 38 cycles. For CX3CR1 [primers described in Muehlhoefer et al. (Muehlhoefer et al., 2000)], amplification References was performed at 94, 55 and 72°C for 30 seconds, 30 seconds and 45 seconds, Allan, L. A., Morrice, N., Brady, S., Magee, G., Pathak, S. and Clarke, P. R. (2003). respectively. For VCAM-1 [primers described in Serradell et al. (Serradell et al., Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat. Cell 2002)], amplification was performed at 94, 53 and 72°C for 30 seconds, 30 seconds Biol. 5, 647-654. and 45 seconds, respectively, for 38 cycles. For 4 integrin (GenBank # Anderson, H. A., Englert, R., Gursel, I. and Shacter, E. (2002). Oxidative stress inhibits NM_000885), the sense primer used was 5-CGA ACC GAT GGC TCC TA-3 and the phagocytosis of apoptotic cells that have externalized phosphatidylserine. Cell the antisense primer was 5-AGT ATG CTG GCT CCG AAA AT-3, amplification Death Differ. 9, 616-625. was performed at 94, 55 and 72°C for 30 seconds, 30 seconds and 45 seconds, Bazan, J. F., Bacon, K. B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D. respectively, for 40 cycles. For ICAM-1 [primers described in Besch et al. (Besch R., Zlotnik, A. and Schall, T. J. (1997). A new class of membrane-bound chemokine et al., 2002)], amplification was performed at 94, 65 and 72°C for 30 seconds, 30 with a CX3C motif. Nature 385, 640-644. seconds and 45 seconds, respectively, for 50 cycles. For L integrin (GenBank # Besch, R., Giovannangeli, C., Kammerbauer, C. and Degitz, K. (2002). Specific BC008777), the sense primer used was 5-TTT GAG AAG AAC TGT GGG GAG inhibition of ICAM-1 expression mediated by gene targeting with Triplex-forming GAC-3 and the antisense primer was 5-GGT GGG CGA GAT GGA AGG T-3, oligonucleotides. J. Biol. Chem. 277, 32473-32479. both amplification was performed at 94, 60 and 72°C for 30 seconds, 45 seconds Bird, I. N., Taylor, V., Newton, J. P., Spragg, J. H., Simmons, D. L., Salmon, M. and and 2 minutes, respectively, for 40 cycles. Amplification products (10 l) were Buckley, C. D. (1999). Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration. J. Cell Sci. 112, 1989- subjected to electrophoresis on 2% agarose gel containing ethidium bromide for visualisation. Blasi, E., Back, T. C., Stull, S. W. and Varesio, L. (1987). Regulation of bone marrow cell survival in short-term cultures: a new macrophage function. Cell Immunol. 104, Immunoblotting 334-342. Total proteins from mpc and MP cultures, and co-cultures of mpcs with MPs, were Boehme, S. A., Lio, F. M., Maciejewski-Lenoir, D., Bacon, K. B. and Conlon, P. J. extracted as described in Davaille et al. (Davaille et al., 2002). Protein concentration (2000). The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia. was determined using the BCA protein assay kit. Aliquots corresponding to 15 g J. Immunol. 165, 397-403. of proteins were subjected to western blot. Anti-phosphorylated Akt (1/1000; Cell Borthwick, N. J., Akbar, A. A., Buckley, C., Pilling, D., Salmon, M., Jewell, A. P. and Signalling Technology), anti-phosphorylated ERK1/2 (1/1000; Promega), anti-Bcl- Yong, K. L. (2003). Transendothelial migration confers a survival advantage to 2 (1/500; Santa Cruz Biotechnology), anti-PECAM-1 (1/500, Dakocytomation) or activated T lymphocytes: role of LFA-1/ICAM-1 interactions. Clin. Exp. Immunol. 134, anti--actin (1/1000; Santa Cruz Biotechnology) antibodies were added overnight 246-252. and revealed using peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat Brand, S., Sakaguchi, T., Gu, X., Colgan, S. P. and Reinecker, H. C. (2002). antibodies (1/4000; Santa Cruz), which was detected using a chemiluminescence Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses kit (Amersham Biosciences). in intestinal epithelial cells. Gastroenterology 122, 166-177. Burgess, D. H., Svensson, M., Dandrea, T., Gronlund, K., Hammarquist, F., Orrenius, S. and Cotgreave, I. A. (1999). Human skeletal muscle cytosols are refractory to In vitro immunolabellings cytochrome c-dependent activation of type-II caspases and lack APAF-1. Cell Death Human cells cultured on coverslips were labelled with primary antibodies Differ. 6, 256-261. (same references as above) for 2 hours: anti-CX3CL1 (50 g/ml), anti-CX3CR1 Cantini, M. and Carraro, U. (1995). Macrophage-released factor stimulates selectively (15 g/ml), anti-VCAM-1 (15 g/ml), anti-VLA-4 (15 g/ml), anti-ICAM-1 myogenic cells in primary muscle culture. J. Neuropathol. Exp. Neurol. 54, 121-128. (15 g/ml), anti-LFA-1 (15 g/ml), anti-PECAM-1 (15 g/ml), revealed using Cantini, M., Massimino, M. L., Bruson, A., Catani, C., Dalla, L. L. and Carraro, U. biotinylated antibody (1/200), HRP-streptavidine (1/200) and DAB substrate (1994). Macrophages regulate proliferation and differentiation of satellite cells. kit for peroxidase (Vector Laboratories). Controls included incubation with Biochem. Biophys. Res. Commun. 202, 1688-1696. whole IgGs from the species of the primary antibody (50 g/ml; Vector Chapman, G. A., Moores, K., Harrison, D., Campbell, C. A., Stewart, B. R. and Laboratories). Strijbos, P. J. (2000). Fractalkine cleavage from neuronal membranes represents an acute event in the inflammatory response to excitotoxic brain damage. J. Neurosci. 20, In vivo toxic muscle injury RC87. Charbord, P. and Moore, K. (2005). Gene expression in stem cell-supporting stromal Notexin (10 l of 25 g/ml in PBS; Sigma) was injected into the tibialis anterior cell lines. Ann. N.Y. Acad. Sci. 1044, 159-167. of adult C57/B6 mice. At various times after injection, muscles were removed, snap Charbord, P., Remy-Martin, J. P., Tamayo, E., Bernard, G., Keating, A. and Peault, frozen in nitrogen-chilled isopentane (–160°C) and kept at –80°C until use. 7 m- B. (2000). Analysis of the microenvironment necessary for engraftment: role of the thick cryosections were treated for immunolabelling. vascular smooth muscle-like stromal cells. J. Hematother. Stem Cell Res. 9, 935-943. Chazaud, B., Hittinger, L., Sonnet, C., Champagne, S., Le Corvoisier, P., Benhaiem- In situ detection of apoptosis (2003a). Sigaux, N., Unterseeh, T., Su, J., Merlet, P., Rahmouni, A. et al. Muscle cryosections were incubated with rabbit polyclonal desmin antibodies (60 Endoventricular porcine autologous myoblast transplantation can be successfully g/ml; Abcam) and further treated to detect apoptotic nuclei (Apoptag Red; achieved with minor mechanical cell damage. Cardiovasc. Res. 58, 444-450. Qbiogen). Slides were examined under an Axioplan 2 Zeiss microscope (Carl Zeiss) Chazaud, B., Sonnet, C., Lafuste, P., Bassez, G., Rimaniol, A. C., Poron, F., Authier, and images were captured with an Orca ER digital camera (Hamamatsu Photonics F. J., Dreyfus, P. A. and Gherardi, R. K. (2003b). Satellite cells attract monocytes KK) using Simple PCI software (C-Imaging Compix). Apoptotic desmin and and use macrophages as a support to escape apoptosis and enhance muscle growth. J. desmin cells were counted in at least 20 randomly chosen fields within the injured Cell Biol. 163, 1133-1143. area (20 objective). Columbaro, M., Mattioli, E., Lattanzi, G., Rutigliano, C., Ognibene, A., Maraldi, N. Journal of Cell Science 2506 Journal of Cell Science 119 (12) M. and Squarzoni, S. (2001). Staurosporine treatment and serum starvation promote and Authier, F. J. (2005). ADAM12 and alpha9beta1 integrin are instrumental in the cleavage of emerin in cultured mouse myoblasts: involvement of a caspase- human myogenic cell differentiation. Mol. Biol. Cell 16, 861-870. dependent mechanism. FEBS Lett. 509, 423-429. Lapidot, T. and Petit, I. (2002). Current understanding of stem cell mobilization: the Davaille, J., Li, L., Mallat, A. and Lotersztajn, S. (2002). Sphingosine 1-phosphate roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal triggers both apoptotic and survival signals for human hepatic myofibroblasts. J. Biol. cells. Exp. Hematol. 30, 973-981. Chem. 277, 37323-37330. Laskin, D. L. and Laskin, J. D. (2001). Role of macrophages and inflammatory Deiva, K., Geeraerts, T., Salim, H., Leclerc, P., Hery, C., Hugel, B., Freyssinet, J. M. mediators in chemically induced toxicity. Toxicology 160, 111-118. and Tardieu, M. (2004). Fractalkine reduces N-methyl-d-aspartate-induced calcium Lawlor, M. A. and Rotwein, P. (2000). Insulin-like growth factor-mediated muscle cell flux and apoptosis in human neurons through extracellular signal-regulated kinase survival: central roles for Akt and cyclin-dependent kinase inhibitor p21. Mol. Cell. activation. Eur. J. Neurosci. 20, 3222-3232. Biol. 20, 8983-8995. Dominov, J. A., Dunn, J. J. and Miller, J. B. (1998). Bcl-2 expression identifies an early Lefaucheur, J. P. and Sebille, A. (1995). The cellular events of injured muscle stage of myogenesis and promotes clonal expansion of muscle cells. J. Cell Biol. 142, regeneration depend on the nature of the injury. Neuromuscul. Disord. 5, 501-509. 537-544. Lescaudron, L., Peltekian, E., Fontaine-Perus, J., Paulin, D., Zampieri, M., Garcia, Du, J., Wang, X., Miereles, C., Bailey, J. L., Debigare, R., Zheng, B., Price, S. R. and L. and Parrish, E. (1999). Blood borne macrophages are essential for the triggering Mitch, W. E. (2004). Activation of caspase-3 is an initial step triggering accelerated of muscle regeneration following muscle transplant. Neuromuscul. Disord. 9, 72-80. muscle proteolysis in catabolic conditions. J. Clin. Invest. 113, 115-123. Leu, S. T., Jacques, S. A., Wingerd, K. L., Hikita, S. T., Tolhurst, E. C., Pring, J. L., Elliott, J. A. and Winn, W. C., Jr (1986). Treatment of alveolar macrophages with Wiswell, D., Kinney, L., Goodman, N. L., Jackson, D. Y. et al. (2004). Integrin cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. alpha4beta1 function is required for cell survival in developing retina. Dev. Biol. 276, Infect. Immun. 51, 31-36. 416-430. Evans, P. C., Taylor, E. R. and Kilshaw, P. J. (2001). Signaling through CD31 protects Leussink, V. I., Zettl, U. K., Jander, S., Pepinsky, R. B., Lobb, R. R., Stoll, G., Toyka, endothelial cells from apoptosis. Transplantation 71, 457-460. K. V. and Gold, R. (2002). Blockade of signaling via the very late antigen (VLA-4) Ferrero, E., Belloni, D., Contini, P., Foglieni, C., Ferrero, M. E., Fabbri, M., Poggi, and its counterligand vascular cell adhesion molecule-1 (VCAM-1) causes increased A. and Zocchi, M. R. (2003). Transendothelial migration leads to protection from T cell apoptosis in experimental autoimmune neuritis. Acta Neuropathol. 103, 131-136. starvation-induced apoptosis in CD34+CD14+ circulating precursors: evidence for Lucas, A. D., Chadwick, N., Warren, B. F., Jewell, D. P., Gordon, S., Powrie, F. and PECAM-1 involvement through Akt/PKB activation. Blood 101, 186-193. Greaves, D. R. (2001). The transmembrane form of the CX3CL1 chemokine Fong, A. M., Robinson, L. A., Steeber, D. A., Tedder, T. F., Yoshie, O., Imai, T. and fractalkine is expressed predominantly by epithelial cells in vivo. Am. J. Pathol. 158, Patel, D. D. (1998). Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte 855-866. capture, firm adhesion, and activation under physiologic flow. J. Exp. Med. 188, 1413- Massimino, M. L., Rapizzi, E., Cantini, M., Libera, L. D., Mazzoleni, F., Arslan, P. 1419. and Carraro, U. (1997). ED2+ macrophages increase selectively myoblast Gao, C., Sun, W., Christofidou-Solomidou, M., Sawada, M., Newman, D. K., Bergom, proliferation in muscle cultures. Biochem. Biophys. Res. Commun. 235, 754-759. C., Albelda, S. M., Matsuyama, S. and Newman, P. J. (2003). PECAM-1 functions Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, as a specific and potent inhibitor of mitochondrial-dependent apoptosis. Blood 102, 493-495. 169-179. McArdle, A., Maglara, A., Appleton, P., Watson, A. J., Grierson, I. and Jackson, M. Garcia-Martinez, V., Macias, D., Ganan, Y., Garcia-Lobo, J. M., Francia, M. V., J. (1999). Apoptosis in multinucleated skeletal muscle myotubes. Lab. Invest. 79, 1069- Fernandez-Teran, M. A. and Hurle, J. M. (1993). Internucleosomal DNA 1076. fragmentation and programmed cell death (apoptosis) in the interdigital tissue of the McClearn, D., Medville, R. and Noden, D. (1995). Muscle cell death during the embryonic chick leg bud. J. Cell Sci. 106, 201-208. development of head and neck muscles in the chick embryo. Dev. Dyn. 202, 365-377. Geske, F. J., Monks, J., Lehman, L. and Fadok, V. A. (2002). The role of McLennan, I. S. (1996). Degenerating and regenerating skeletal muscles contain several the macrophage in apoptosis: hunter, gatherer, and regulator. Int. J. Hematol. 76, 16- subpopulations of macrophages with distinct spatial and temporal distributions. J. Anat. 26. 188, 17-28. Goda, S., Imai, T., Yoshie, O., Yoneda, O., Inoue, H., Nagano, Y., Okazaki, T., Imai, Merly, F., Lescaudron, L., Rouaud, T., Crossin, F. and Gardahaut, M. F. (1999). H., Bloom, E. T., Domae, N. et al. (2000). CX3C-chemokine, fractalkine-enhanced Macrophages enhance muscle satellite cell proliferation and delay their differentiation. adhesion of THP-1 cells to endothelial cells through integrin-dependent and Muscle Nerve 22, 724-732. -independent mechanisms. J. Immunol. 164, 4313-4320. Meucci, O., Fatatis, A., Simen, A. A. and Miller, R. J. (2000). Expression of CX3CR1 Gordon, S. (1995). The macrophage. BioEssays 17, 977-986. chemokine receptors on neurons and their role in neuronal survival. Proc. Natl. Acad. Gras, G., Chretien, F., Vallat-Decouvelaere, A. V., Le Pavec, G., Porcheray, F., Sci. USA 97, 8075-8080. Bossuet, C., Leone, C., Mialocq, P., Dereuddre-Bosquet, N., Clayette, P. et al. Meyer, D. M., Dustin, M. L. and Carron, C. P. (1995). Characterization of intercellular (2003). Regulated expression of sodium-dependent glutamate transporters and adhesion molecule-1 ectodomain (sICAM-1) as an inhibitor of lymphocyte function- synthetase: a neuroprotective role for activated microglia and macrophages in HIV associated molecule-1 interaction with ICAM-1. J. Immunol. 155, 3578-3584. infection? Brain Pathol. 13, 211-222. Mierke, C. T., Ballmaier, M., Werner, U., Manns, M. P., Welte, K. and Bischoff, S. Grounds, M. D. (1987). Phagocytosis of necrotic muscle in muscle isografts is influenced C. (2000). Human endothelial cells regulate survival and proliferation of human mast by the strain, age, and sex of host mice. J. Pathol. 153, 71-82. cells. J. Exp. Med. 192, 801-811. Hall, B. M., Fortney, J. E., Taylor, L., Wood, H., Wang, L., Adams, S., Davis, S. and Minges Wols, H. A., Underhill, G. H., Kansas, G. S. and Witte, P. L. (2002). The role Gibson, L. F. (2004). Stromal cells expressing elevated VCAM-1 enhance survival of of bone marrow-derived stromal cells in the maintenance of plasma cell longevity. J. B lineage tumor cells. Cancer Lett. 207, 229-239. Immunol. 169, 4213-4221. Hanspal, M. and Hanspal, J. S. (1994). The association of erythroblasts with Mishra, D. K., Friden, J., Schmitz, M. C. and Lieber, R. L. (1995). Anti-inflammatory macrophages promotes erythroid proliferation and maturation: a 30-kD heparin- medication after muscle injury. A treatment resulting in short-term improvement but binding protein is involved in this contact. Blood 84, 3494-3504. subsequent loss of muscle function. J. Bone Joint Surg. Am. 77, 1510-1519. Harrison, J. K., Jiang, Y., Chen, S., Xia, Y., Maciejewski, D., McNamara, R. K., Miyagoe, Y., Hanaoka, K., Nonaka, I., Hayasaka, M., Nabeshima, Y., Arahata, K., Streit, W. J., Salafranca, M. N., Adhikari, S., Thompson, D. A. et al. (1998). Role Nabeshima, Y. and Takeda, S. (1997). Laminin alpha2 chain-null mutant mice by for neuronally derived fractalkine in mediating interactions between neurons and targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient CX3CR1-expressing microglia. Proc. Natl. Acad. Sci. USA 95, 10896-10901. congenital muscular dystrophy. FEBS Lett. 415, 33-39. Hawke, T. J. and Garry, D. J. (2001). Myogenic satellite cells: physiology to molecular Mizuno, T., Kawanokuchi, J., Numata, K. and Suzumura, A. (2003). Production and biology. J. Appl. Physiol. 91, 534-551. neuroprotective functions of fractalkine in the central nervous system. Brain Res. 979, Hayashida, K., Shimaoka, Y., Ochi, T. and Lipsky, P. E. (2000). Rheumatoid arthritis 65-70. synovial stromal cells inhibit apoptosis and up-regulate Bcl-xL expression by B cells Montanaro, F., Lindenbaum, M. and Carbonetto, S. (1999). alpha-Dystroglycan is a in a CD49/CD29-CD106-dependent mechanism. J. Immunol. 164, 1110-1116. laminin receptor involved in extracellular matrix assembly on myotubes and muscle Huppertz, B., Tews, D. S. and Kaufmann, P. (2001). Apoptosis and syncytial fusion in cell viability. J. Cell Biol. 145, 1325-1340. human placental trophoblast and skeletal muscle. Int. Rev. Cytol. 205, 215-253. Muehlhoefer, A., Saubermann, L. J., Gu, X., Luedtke-Heckenkamp, K., Xavier, R., Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, Blumberg, R. S., Podolsky, D. K., MacDermott, R. P. and Reinecker, H. C. (2000). M., Takagi, S., Nomiyama, H., Schall, T. J. et al. (1997). Identification and molecular Fractalkine is an epithelial and endothelial cell-derived chemoattractant for characterization of fractalkine receptor CX3CR1, which mediates both leukocyte intraepithelial lymphocytes in the small intestinal mucosa. J. Immunol. 164, 3368- migration and adhesion. Cell 91, 521-530. 3376. Jejurikar, S. S. and Kuzon, W. M., Jr (2003). Satellite cell depletion in degenerative Muller-Sieburg, C. E. and Deryugina, E. (1995). The stromal cells’ guide to the stem skeletal muscle. Apoptosis 8, 573-578. cell universe. Stem Cells 13, 477-486. Koopman, G., Keehnen, R. M., Lindhout, E., Newman, W., Shimizu, Y., van Seventer, Nakanishi, K., Sudo, T. and Morishima, N. (2005). Endoplasmic reticulum stress G. A., de Groot, C. and Pals, S. T. (1994). Adhesion through the LFA-1 signaling transmitted by ATF6 mediates apoptosis during muscle development. J. Cell (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) Biol. 169, 555-560. pathways prevents apoptosis of germinal center B cells. J. Immunol. 152, 3760-3767. Napier, J. R., Thomas, M. F., Sharma, M., Hodgkinson, S. C. and Bass, J. J. (1999). Koseki, T., Inohara, N., Chen, S. and Nunez, G. (1998). ARC, an inhibitor of apoptosis Insulin-like growth factor-I protects myoblasts from apoptosis but requires other factors expressed in skeletal muscle and heart that interacts selectively with caspases. Proc. to stimulate proliferation. J. Endocrinol. 163, 63-68. Natl. Acad. Sci. USA 95, 5156-5160. Oostendorp, R. A., Reisbach, G., Spitzer, E., Thalmeier, K., Dienemann, H., Lafuste, P., Sonnet, C., Chazaud, B., Dreyfus, P. A., Gherardi, R. K., Wewer, U. M. Mergenthaler, H. G. and Dormer, P. (1995). VLA-4 and VCAM-1 are the principal Journal of Cell Science Macrophages and myogenic cell survival 2507 adhesion molecules involved in the interaction between blast colony-forming cells and Song, G., Ouyang, G. and Bao, S. (2005). The activation of Akt/PKB signaling pathway bone marrow stromal cells. Br. J. Haematol. 91, 275-284. and cell survival. J. Cell Mol. Med. 9, 59-71. Ostrovsky, O. and Bengal, E. (2003). The mitogen-activated protein kinase cascade Takeishi, T., Hirano, K., Kobayashi, T., Hasegawa, G., Hatakeyama, K. and Naito, promotes myoblast cell survival by stabilizing the cyclin-dependent kinase inhibitor, M. (1999). The role of Kupffer cells in liver regeneration. Arch. Histol. Cytol. 62, 413- p21WAF1 protein. J. Biol. Chem. 278, 21221-21231. 422. Pimorady-Esfahani, A., Grounds, M. D. and McMenamin, P. G. (1997). Macrophages Taverna, D., Disatnik, M. H., Rayburn, H., Bronson, R. T., Yang, J., Rando, T. A. and dendritic cells in normal and regenerating murine skeletal muscle. Muscle Nerve and Hynes, R. O. (1998). Dystrophic muscle in mice chimeric for expression of alpha5 20, 158-166. integrin. J. Cell Biol. 143, 849-859. Polazzi, E., Gianni, T. and Contestabile, A. (2001). Microglial cells protect cerebellar Tews, D. S. (2002). Apoptosis and muscle fibre loss in neuromuscular disorders. granule neurons from apoptosis: evidence for reciprocal signaling. Glia 36, 271-280. Neuromuscul. Disord. 12, 613-622. Rehfeldt, C., Renne, U., Wittstock, M., Mix, E. and Zettl, U. K. (2004). Long-term Tidball, J. G., Albrecht, D. E., Lokensgard, B. E. and Spencer, M. J. (1995). Apoptosis growth selection of mice changes the intrinsic susceptibility of myogenic cells to precedes necrosis of dystrophin-deficient muscle. J. Cell Sci. 108, 2197-2204. apoptosis. J. Muscle Res. Cell Motil. 25, 177-185. Tureckova, J., Wilson, E. M., Cappalonga, J. L. and Rotwein, P. (2001). Insulin-like Reuveny, M., Heller, H. and Bengal, E. (2004). RhoA controls myoblast survival by growth factor-mediated muscle differentiation: collaboration between inducing the phosphatidylinositol 3-kinase-Akt signaling pathway. FEBS Lett. 569, phosphatidylinositol 3-kinase-Akt-signaling pathways and myogenin. J. Biol. Chem. 129-134. 276, 39264-39270. Robertson, T. A., Maley, M. A., Grounds, M. D. and Papadimitriou, J. M. (1993). Umehara, H., Goda, S., Imai, T., Nagano, Y., Minami, Y., Tanaka, Y., Okazaki, T., The role of macrophages in skeletal muscle regeneration with particular reference to Bloom, E. T. and Domae, N. (2001). Fractalkine, a CX3C-chemokine, functions chemotaxis. Exp. Cell Res. 207, 321-331. predominantly as an adhesion molecule in monocytic cell line THP-1. Immunol. Cell Rose, D. M., Cardarelli, P. M., Cobb, R. R. and Ginsberg, M. H. (2000). Soluble Biol. 79, 298-302. VCAM-1 binding to alpha4 integrins is cell-type specific and activation dependent and Vachon, P. H., Loechel, F., Xu, H., Wewer, U. M. and Engvall, E. (1996). Merosin and is disrupted during apoptosis in T cells. Blood 95, 602-609. laminin in myogenesis; specific requirement for merosin in myotube stability and Rosen, G. D., Sanes, J. R., LaChance, R., Cunningham, J. M., Roman, J. and Dean, survival. J. Cell Biol. 134, 1483-1497. D. C. (1992). Roles for the integrin VLA-4 and its counter receptor VCAM-1 in Vachon, P. H., Xu, H., Liu, L., Loechel, F., Hayashi, Y., Arahata, K., Reed, J. C., myogenesis. Cell 69, 1107-1119. Wewer, U. M. and Engvall, E. (1997). Integrins (alpha7beta1) in muscle function and Rubartelli, A., Poggi, A. and Zocchi, M. R. (1997). The selective engulfment of survival. Disrupted expression in merosin-deficient congenital muscular dystrophy. J. apoptotic bodies by dendritic cells is mediated by the alpha(v)beta3 integrin and Clin. Invest. 100, 1870-1881. requires intracellular and extracellular calcium. Eur. J. Immunol. 27, 1893-1900. Wang, J. and Walsh, K. (1996). Resistance to apoptosis conferred by Cdk inhibitors Ruest, L. B., Khalyfa, A. and Wang, E. (2002). Development-dependent disappearance during myocyte differentiation. Science 273, 359-361. of caspase-3 in skeletal muscle is post-transcriptionally regulated. J. Cell Biochem. 86, Wang, J. and Lenardo, M. J. (1997). Essential lymphocyte function associated 1 (LFA- 21-28. 1): intercellular adhesion molecule interactions for T cell-mediated B cell apoptosis by Sadahira, Y. and Mori, M. (1999). Role of the macrophage in erythropoiesis. Pathol. Fas/APO-1/CD95. J. Exp. Med. 186, 1171-1176. Int. 49, 841-848. Wang, M. W., Consoli, U., Lane, C. M., Durett, A., Lauppe, M. J., Champlin, R., Schwartz, M. A. and Ingber, D. E. (1994). Integrating with integrins. Mol. Biol. Cell 5, Andreeff, M. and Deisseroth, A. B. (1998). Rescue from apoptosis in early (CD34- 389-393. selected) versus late (non-CD34-selected) human hematopoietic cells by very late Serradell, M., Diaz-Ricart, M., Cases, A., Zurbano, M. J., Lopez-Pedret, J., Arranz, antigen 4- and vascular cell adhesion molecule (VCAM) 1-dependent adhesion to bone O., Ordinas, A. and Escolar, G. (2002). Uremic medium causes expression, marrow stromal cells. Cell Growth Differ. 9, 105-112. redistribution and shedding of adhesion molecules in cultured endothelial cells. Winter, S. S., Sweatman, J. J., Lawrence, M. B., Rhoades, T. H., Hart, A. L. and Haematologica 87, 1053-1061. Larson, R. S. (2001). Enhanced T-lineage acute lymphoblastic leukaemia cell survival Shen, W., Li, Y., Tang, Y., Cummins, J. and Huard, J. (2005). NS-398, a on bone marrow stroma requires involvement of LFA-1 and ICAM-1. Br. J. Haematol. cyclooxygenase-2-specific inhibitor, delays skeletal muscle healing by decreasing 115, 862-871. regeneration and promoting fibrosis. Am. J. Pathol. 167, 1105-1117. Zen, K., Masuda, J. and Ogata, J. (1996). Monocyte-derived macrophages prime Skuk, D. and Tremblay, J. P. (2000). Progress in myoblast transplantation: a potential peripheral T cells to undergo apoptosis by cell-cell contact via ICAM-1/LFA-1- treatment of dystrophies. Microsc. Res. Tech. 48, 213-222. dependent mechanism. Immunobiology 195, 323-333. 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Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems

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Abstract

Research Article 2497 Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems Corinne Sonnet*, Peggy Lafuste*, Ludovic Arnold, Madly Brigitte, Françoise Poron, François-Jérôme Authier, Fabrice Chrétien, Romain K. Gherardi and Bénédicte Chazaud INSERM E0011 ‘Cellular interactions in the neuromuscular system’, Faculté de Médecine, Institut National de la Santé et de la Recherche Médicale; Université Paris XII, 8 rue du Général Sarrail, 94000 Créteil, France *These authors contributed equally to this work Author for correspondence (e-mail: [email protected]) Accepted 22 March 2006 Journal of Cell Science 119, 2497-2507 Published by The Company of Biologists 2006 doi:10.1242/jcs.02988 Summary The mechanisms underlying stromal cell supportive survival pathways; (3) four pro-survival cell-cell adhesion functions are incompletely understood but probably molecular systems detected by DNA macroarray are implicate a mixture of cytokines, matrix components and expressed by macrophages and myogenic cells in vitro and cell adhesion molecules. Skeletal muscle uses recruited in vivo – VCAM-1–VLA-4, ICAM-1–LFA-1, PECAM- macrophages to support post-injury regeneration. We and 1–PECAM-1 and CX3CL1-CX3CR1; (4) macrophages others have previously shown that macrophages secrete deliver anti-apoptotic signals through all four adhesion mitogenic factors for myogenic cells. Here, we focused on systems, as assessed by functional analyses with blocking macrophage-elicited survival signals. We demonstrated antibodies; and (5) macrophages more strongly rescue that: (1) macrophage influx is temporally correlated with differentiated myotubes, which must achieve adhesion- the disappearance of TUNEL-positive apoptotic myogenic induced stabilisation of their structure to survive. cells during post-injury muscle regeneration in mice; (2) Macrophages could secure these cells until they establish direct cell-cell contacts between human macrophages and final association with the matrix. myogenic cells rescue myogenic cells from apoptosis, as assessed by decreased annexin V labelling and caspase-3 activity, and by increased DIOC-6 staining, Bcl-2 Key words: Skeletal muscle, Myogenic precursor cells, Macrophage, expression and phosphorylation of Akt and ERK1/2 Apoptosis, Cell adhesion molecules Introduction components and cell adhesion molecules (Charbord and Stromal cells are microenvironmental cells defined by their Moore, 2005). ability to support the development, maintenance, proliferation The spectrum of stromal cells is debated and remains ill and differentiation of tissue-specific cell types. For example, defined (Muller-Sieburg and Deryugina, 1995). Nevertheless, supportive stromal cells are crucially involved in macrophages (MPs) constitute one definite cell type in this haematopoiesis. The supportive stromal cell compartment is spectrum and were previously recognised to play a major role established before the formation of the haematopoietic system; in tissue repair and homeostasis maintenance (Gordon, 1995). it remains dynamic, instead of being static once established, In addition to their classical functions, including microbicidal reacting to extrinsic signals that can either damage or enhance activity, phagocytosis and antigen presentation, these stromal cell function and number (Muller-Sieburg and multifaceted cells efficiently support growth and differentiation Deryugina, 1995). In non-haematopoietic tissues, organogenic of other cell types (Gordon, 1995; Laskin and Laskin, 2001). processes are similarly driven by stromal cells that control Their supportive effect was documented with respect to parenchymal cell functions such as migration, proliferation, erythroblasts, hepatocytes, neurons, oligodendrocytes and differentiation and programmed cell death (Charbord et al., myogenic cells (Blasi et al., 1987; Cantini et al., 1994; 2000; Lapidot and Petit, 2002). Sadahira and Mori, 1999; Takeishi et al., 1999; Polazzi et al., The mechanisms underlying supportive functions of 2001; Gras et al., 2003; Chazaud et al., 2003b). stromal cells are incompletely understood. Stromal cells In contrast to bone marrow, where stromal cells are in place probably provide a complex molecular milieu that influences to support an ever-changing haematopoietic compartment, the behaviour of local stem cells, which have the choice skeletal muscle is normally a stable tissue that uses newly of many fates, including quiescence, self-renewal, recruited MPs to support post-injury muscle regeneration differentiation and apoptosis (Charbord and Moore, 2005). (McLennan, 1996; Pimorady-Esfahani et al., 1997; Lescaudron The molecules in this milieu are not well defined but probably et al., 1999). In a previous study, we found that, upon include a mixture of cytokines, extracellular matrix activation, a small myogenic stem cell population residing Journal of Cell Science 2498 Journal of Cell Science 119 (12) beneath the basal lamina of each adult myofibre, the so-called molecule 1 (ICAM-1; CD54) binding to leukocyte function muscle satellite cells (Mauro, 1961), can attract circulating associated molecule 1 (LFA-1); chemokine CX3CL1 binding monocytes and interplay with MPs to enhance their growth to CX3CR1; and platelet-endothelial cell adhesion molecule 1 (Chazaud et al., 2003b). In vitro studies suggested that MPs (PECAM-1; CD31) homophilic binding to another PECAM-1. can support myogenic precursor cell (mpc) growth by Finally, we used a mouse model of post-injury muscle stimulating their proliferation through soluble mitogenic regeneration to demonstrate spatiotemporal correlation factors, and by preventing their apoptosis through direct cell- between MP influx and fading of injury-induced mpc cell contacts involving unknown molecular systems (Chazaud apoptosis. et al., 2003b). MP-derived soluble factors inducing mpc proliferation have Results long been reported (Cantini et al., 1994; Cantini and Carraro, MPs inhibit both spontaneous and induced mpc 1995; Massimino et al., 1997), and the literature on myogenic apoptosis in a dose-dependent way cell growth factors is extensive (reviewed by Hawke and Garry, As assessed by annexin V labelling, the addition of human 2001). By contrast, the significance of direct contacts between monocyte-derived MPs to primary mpc cultures inhibited MPs and mpcs has not been previously explored in the setting spontaneous apoptosis of mononucleated myoblasts (Fig. 1A). of muscle regeneration. In fact, relatively little is known The protective effect of MPs was dose dependent (P<0.05), regarding the relevance of apoptosis to skeletal muscle apoptosis being inhibited by 75% at the 1:5 (mpc:MP) ratio homeostasis and repair, although evidence exists indicating (P<0.001) (Fig. 1A). Because the rate of spontaneous mpc that enhanced apoptosis plays a role during muscle aging, apoptosis was low (7.1±1.3% of the cells), further experiments muscular dystrophy, muscle denervation and unloading were performed after induction of mpc apoptosis by STS (reviewed by Jejurikar and Kuzon, 2003). Normal adult (Dominov et al., 1998; Columbaro et al., 2001). The protective myofibres are somewhat resistant to apoptosis. Their effect of MPs was strong enough to reduce STS-induced mpc sarcoplasm is refractory to mitochondrial cytochrome c- apoptosis (Fig. 1B). At the 1:10 (mpc:MP) ratio, apoptosis was dependent activation of type II caspases (Burgess et al., 1999). inhibited by 74% as assessed by annexin V labelling (P<0.001) Caspase-3 protein, which acts on the execution of cell death, (Fig. 1B) and by 60% as assessed by DIOC-6 staining is absent in normal myofibres (Ruest et al., 2002). Upstream (P<0.01). MPs inhibited STS-induced myoblast apoptosis in a protective mechanisms against apoptosis include blockage of dose-dependent and saturable way (P<0.001) (Fig. 1B). the two caspase-3 activation pathways, as the caspase-8 inhibitor ARC is expressed (Koseki et al., 1998), and caspase- The anti-apoptotic effect of MPs is more pronounced in 9 activator Apaf-1 is absent (Burgess et al., 1999), from myotubes skeletal muscle. The only physiological circumstance in which In culture, mpcs proliferate and give rise to mononucleated caspase-3 protein appears in adults is in regenerating muscle myoblasts that subsequently fuse with each other to form (Ruest et al., 2002). Such an expression of caspase-3 protein multinucleated myotubes. It is well established that myoblast in regenerating muscle is transient and might allow muscle to apoptosis occurs at times of serum deprivation used to boost get rid of excess replicating satellite cells or to delete myogenic differentiation. In addition, it has been shown that improperly innervated, newly formed myofibres (Ruest et al., myotubes are at particular risk of undergoing apoptosis upon 2002). In addition to its role in apoptosis, caspase-3 also stimulation with extrinsic stimuli as a result of their poor Bcl- participates to myofibrillar proteolysis (Du et al., 2004). Once 2 expression (Dominov et al., 1998; McArdle et al., 1999; regeneration is complete, caspase-3 mRNA remains detectable Ruest et al., 2002). Consistently, in our experiments, myotubes in the repaired muscle whereas caspase-3 protein becomes were 1.6-fold more sensitive to STS than myoblasts (P<0.05) undetectable (Ruest et al., 2002). Finally, from an evolutionary (Fig. 1C,D). Because the large size of myotubes precluded flow perspective, it seems important for skeletal muscle tissue to be cytometry analysis, determination of caspase-3 activity was protected from pro-apoptotic signals linked to exercise- used to compare the anti-apoptotic effect of MPs on STS- associated mitochondrial stress (Burgess et al., 1999) and, treated myoblasts and myotubes (Dominov et al., 1998). MPs consequently, mechanisms promoting restoration of the more efficiently rescued myotubes than myoblasts from STS- protected status of myogenic cells after muscle damage must induced apoptosis, as they decreased caspase-3 activity by 21% exist and could implicate stromal cells. in myoblasts and by 39% in myotubes [values at 5 hours, 1:2 We examined if and how MPs could play a significant role (mpc:MP) ratio, P<0.005] (Fig. 1C,D). in regulation of myogenic cell death during regeneration. We first extended our previous observations by analysing MP The anti-apoptotic effect of MPs is associated with protective effects against spontaneous and staurosporine activation of survival signalling (STS)-induced apoptosis of human mononucleated myoblasts Expression of the Bcl-2 anti-apoptotic protein is important for and multinucleated myotubes. Then, we selected candidate survival of expanding myogenic cells (Dominov et al., 1998). anti-apoptotic effector-counterligand molecular systems using As compared with mpc and MP cultures, co-cultures of mpcs DNA macroarray analysis, with confirmatory RT-PCR and with MPs showed enhanced expression of Bcl-2 (Fig. 2A). Pro- immunodetection in human MPs and mpcs. Four systems survival signalling pathways in myogenic cells include the previously implicated in cell-contact-mediated survival of mitogen-activated protein kinase and extracellular signal- other cell types were identified and shown to mediate in vitro regulated kinase (MAPK-ERK1/2) cascade, and the MP anti-apoptotic effects on mpcs by functional studies: phosphatidylinositol 3-kinase (PI 3-kinase) and serine/ vascular cell adhesion molecule 1 (VCAM-1; CD106) binding threonine protein kinase Akt/PKB pathway (Ostrovsky and to very late antigen 4 (VLA-4); intercellular cell adhesion Bengal, 2003; Reuveny et al., 2004). These pathways operate Journal of Cell Science Macrophages and myogenic cell survival 2499 Fig. 2. Induction of pro-survival signals in co-cultures Fig. 1. Inhibition of both spontaneous and induced mpc apoptosis by MPs. of mpcs with MPs. (A) Examples of immunoblots of (A,B) untreated (A) and STS-treated (B) mpcs were co-cultured with MPs at Bcl-2, phosphorylated Akt (Akt-P) and phosphorylated various ratios and mpc apoptosis was evaluated by annexin V labelling (white ERK1/2 (ERK1/2-P) in myoblasts (Mb), myotubes bars) and DIOC-6 staining (black bars) after exclusion of CD14 cells. In A, all (MT), MP cultures and co-cultures. Detection of -actin mpc:MP ratios used were statistically different from mpcs (1:0 ratio) (P0.04); was used to check the protein amount deposited in each in B, all mpc:MP ratios used, except the 1:0.5 ratio, were statistically different well. (B) Apoptosis of mpcs was detected with annexin from mpcs (1:0 ratio) (P0.07). (C,D) STS-treated myoblasts (C, Mb) and V (white bars) and DIOC-6 (black bars) in co-cultures myotubes (D, MT) were co-cultured with or without MP (1:2 mpc:MP ratio). of mpcs with MPs in the presence of H O at low 2 2 Apoptosis was evaluated by caspase-3 activity measurement. Results are means concentration (0.2 mM) or cytochalasin D (1 g/ml). ± s.e.m. of at least three experiments. Results are means ± s.e.m. of three experiments. through sequential phosphorylation events. Both pathways phosphokinase level determination [9.04±4.4 UI/ml in mpc were activated in co-cultures, as assessed by increased culture versus 7.3±3.8 UI/ml in co-cultures of mpcs with MPs phosphorylation of both ERK1/2 and Akt (Fig. 2A). (1:2)]. Altogether, these results substantiate the view that MPs To evaluate to what extent MP phagocytosis of damaged induce pro-survival signalling and decrease mpc apoptosis. cells (Geske et al., 2002) could have participated in the decreased number of apoptotic cells in co-cultures, we used DNA array in MPs and mpcs allows identification of four potent inhibitors of MP phagocytic activity, including H O at anti-apoptotic systems 2 2 low concentrations and cytochalasin D (Elliott and Winn, To select the cell-cell molecular systems at work in the 1986; Rubartelli et al., 1997; Anderson et al., 2002). The transduction of anti-apoptotic signals in mpcs, we used an addition of phagocytosis inhibitors to co-cultures did not mRNA profiling technique that allows analysis of a huge significantly modify the decreased rate of apoptotic mpcs number of genes at once. Among the 375 genes represented on observed in the presence of MPs (Fig. 2B). Consistently, the the DNA macroarray membrane we used, 12 (Table 1) had total number of mpcs did not significantly vary during the 6 products known to be involved in anti-apoptotic signals hour time of co-culture, as assessed both by cell count mediated by cell-cell contacts. [35,400±600 cells/cm in mpc culture versus 35,750±1300 Four of these products were constitutively expressed by cells/cm in co-cultures of mpcs with MPs (1:2)] and creatine human mpcs and had their counterligands expressed by human Journal of Cell Science 2500 Journal of Cell Science 119 (12) Table 1. Gene expression by human mpcs and MPs Intensity* GenBank mpcs stimulated by Expressed protein accession number mpcs MPs MPs versus mpcs Cadherin 5 (VE-cadherin) (homophilic) X79981 ND 304 PECAM-1 (CD31) (homophilic) M28526 106 602 1.5 ALCAM (CD166) (ligand) L38608 204 687 CD6 (receptor) U34625 ND 221 Fractalkine (CX3CL1) (ligand) U91835 90 89 CX3CR1 (receptor) U20350 57 14 1.2 VCAM-1 (CD106) (ligand) X53051 160 162 VLA-44 (CD49d) (receptor subunit) X16983 74 355 1.8 VLA-41 (CD29) (receptor subunit) X07979 464 929 1.2 ICAM-1 (CD54) (ligand) J03132 160 162 LFA-1L (CD11a) (receptor subunit) Y00796 74 355 2.2 LFA-12 (CD18) (receptor subunit) M15395 464 929 1.6 ND: not detected. *Arbitrary units. Intensity detected in mpcs stimulated by MPs (24 hours incubation in MP-conditioned medium as described in Chazaud et al., 2003b) versus intensity detected in mpcs. MPs (Table 1), as follows: (1) VCAM-1 binding to VLA-4 except PECAM-1, which could not be visualised in mpcs (41 integrin); this adhesion system mediates the protective despite positive detection by immunoblotting in differentiated effects of MPs to erythroblasts (Hanspal and Hanspal, 1994; mpcs (Fig. 3B). Expression of all four receptors was much Sadahira and Mori, 1999), of stromal cells to haematopoietic stronger in myotubes than in myoblasts (Fig. 3B). stem cells, B cells and plasma cells (Koopman et al., 1994; Oostendorp et al., 1995; Wang et al., 1998; Hayashida et al., CX3CL1-CX3CR1, VCAM-1–VLA-4, ICAM-1–LFA-1 and 2000; Minges Wols et al., 2002; Hall et al., 2004) and of PECAM-1–PECAM-1 are functional at the mpc surface endothelial cells to mast cells (Mierke et al., 2000). It is also Adhesion assays were used to assess functional availability of involved in protection of T cells and retinal ganglion cells the candidate molecular systems at the MP-mpc interface. It (Rose et al., 2000; Leussink et al., 2002; Leu et al., 2004). (2) was found that mpcs adhered on a MP monolayer in a dose- ICAM-1 binding to LFA-1 (L2 integrin); this system dependent, time-dependent and saturable fashion (Fig. 4A,B), mediates protective effects of endothelial cells to allowing the involvement of specific receptor-ligand transmigrating lymphocytes (Borthwick et al., 2003). Its interactions to be assessed. Blocking antibodies against both implication in support of bone marrow stromal cells to T cells CX3CL1-CX3CR1, VCAM-1–VLA-4, ICAM-1–LFA-1 or (Winter et al., 2001), and of follicular dendritic cells to B cells PECAM-1 failed to inhibit mpc adhesion on MPs significantly (Koopman et al., 1994) has been also reported, but in vitro (data not shown), suggesting robust redundancy of cell experiments have yielded somewhat discrepant results (Zen et adhesion systems involved in mpc-MP contacts. Therefore, al., 1996; Wang and Lenardo, 1997). (3) CX3CL1 (fractalkine) adhesion assays using mpcs deposited on coats of immobilised binding to CX3CR1; CX3CR1 uniquely binds membrane- ligands were used to test the functionality of each receptor. It anchored and shed soluble forms of CX3CL1 (Bazan et al., was shown that mpcs adhered in a dose-dependent and 1997), which are respectively involved in firm cell-to-cell saturable way to VCAM-1 (Fig. 4C), CX3CL1 (Fig. 4D), adhesion (Fong et al., 1998; Umehara et al., 2001) and in ICAM-1 (Fig. 4E) and PECAM-1 (Fig. 4F), as previously chemotaxis (Chapman et al., 2000). MPs and neural cells shown for other cell types (Meyer et al., 1995; Imai et al., 1997; reciprocally signal through this system to suppress apoptotic Bird et al., 1999; Goda et al., 2000). These results strongly cell death (Harrison et al., 1998; Boehme et al., 2000; Meucci suggested that VLA-4, CX3CR1, LFA-1 and PECAM-1 et al., 2000; Mizuno et al., 2003; Deiva et al., 2004). This expressed at the mpc surface were used to bind VCAM-1, system also prevents apoptosis in intestinal epithelium (Brand membrane-bound CX3CL1, ICAM-1 and PECAM-1, et al., 2002). (4) Homophilic PECAM-1 interactions; respectively. endothelial cell PECAM-1 prevents apoptosis of both neighbouring endothelial cells (Bird et al., 1999; Evans et al., CX3CL1-CX3CR1, VCAM-1–VLA-4, ICAM-1–LFA-1 and 2001; Gao et al., 2003) and transmigrating leucocytes (Ferrero PECAM-1–PECAM-1 mediate MP anti-apoptotic activity et al., 2003) through homophilic PECAM-1 interactions. on mpcs Exposure to MP-conditioned medium reinforced mRNA Co-cultures of mpcs with MPs were incubated with blocking expression of all counterreceptors by mpcs (Table 1). Results antibodies to assess anti-apoptotic effects of the selected of DNA macroarray were confirmed by RT-PCR. VCAM-1, molecules in our model. Blockage of each molecular system ICAM-1, CX3CL1 and PECAM-1 mRNAs were detected in inhibited the beneficial effects of MPs on mpc survival MPs (Fig. 3A). It was shown that mpcs, which are already (P<0.05) (Fig. 5), as assessed by increased annexin V labelling known to express the counterreceptor 1 integrin (Vachon et and caspase-3 activity, and decreased DIOC-6 staining al., 1997), expressed 4, L and 2 integrins, and CX3CR1 following exposure to blocking antibodies. Consistently, and PECAM-1 mRNAs (Fig. 3A). The corresponding proteins caspase-3 activity was increased in myotubes in the presence were immunodetected at the cell surface of MPs and mpcs, of the different blocking antibodies (Fig. 5C). Journal of Cell Science Macrophages and myogenic cell survival 2501 Influx of MPs and fading of mpc apoptosis are (Lefaucheur and Sebille, 1995). At 3 hours post-injury, synchronous during post-injury muscle regeneration TUNEL-positive nuclei were abundant in damaged areas (Fig. To examine the relevance of in vitro results, we injected snake 6A) (15.6 apoptotic cells/field). It was found that 60% (9.5 venom notexin in tibialis anterior muscle of adult mice cells/field) of TUNEL-positive cells were desmin negative and presumably corresponded to non-muscle cells; 40% (6.1 cells/field) of apoptotic cells were desmin positive and probably corresponded to activated satellite cells, interstitial myoblasts or myotubes (Fig. 6A). As MP influx proceeded in the regenerating muscle, the number of both the overall apoptotic cell population and of TUNEL-desmin double- positive cells dramatically decreased (Fig. 6A). At 48 hours post-injection, MP infiltration was massive and apoptotic mpcs were extremely rare, accounting for no more than 20% (0.12 cells/field) of the remaining apoptotic cells (0.62 cells/field) at this time (Fig. 6A). At this time point, both anti-apoptotic effectors VCAM-1, ICAM-1, CX3CL1 and PECAM-1, and their counterreceptors VLA-4, LFA-1, CX3CR1 and PECAM- 1, were expressed by MPs and mpcs, respectively, in the regenerating areas (Fig. 6B). Fig. 3. Expression of candidate effectors by human MPs and mpcs. (A) RT-PCR analysis of CX3CL1, VCAM-1, ICAM-1 and PECAM- 1 mRNA in MPs, and of CX3CR1, 4, L, 2 integrins, and PECAM-1 mRNA in mpcs. 2M is beta2microglobulin. Fig. 4. Functionality of candidate effectors at the mpc cell (B) Immunolabelling of CX3CL1, VCAM-1, ICAM-1 and membrane; adhesion assays. (A,B) Adhesion of mpcs on a MP PECAM-1 on MPs (left panel) and of CX3CR1, VLA-4 and LFA-1 monolayer according to incubation time (A) and mpc concentration on mpcs (right panel), revealed by DAB substrate kit for peroxidase. (B). Adhesion of mpcs on VCAM-1 (C), CX3CL1 (D), ICAM-1 (E) Magnification, 300. PECAM-1 expression in mpcs was assessed by and PECAM-1 (F) coats. Results are means ± s.e.m. of three immunoblotting. MT, myotube; Mb, myoblast. experiments. Journal of Cell Science 2502 Journal of Cell Science 119 (12) Discussion Muscle damage is known to induce massive MP infiltration In the present study, we demonstrate that: (1) MP influx is of the injury site (McLennan, 1996; Pimorady-Esfahani et al., temporally correlated with fading of mpc apoptosis during in 1997). Initially, the role of these blood-borne cells was believed vivo post-injury muscle regeneration; (2) MPs rescue to be limited to clearance of necrotic fibres (McLennan, 1996; differentiating mpcs more than cycling mpcs from apoptotic Pimorady-Esfahani et al., 1997). However, several in vivo and cell death; (3) MPs deliver anti-apoptotic signals to mpcs in vitro studies have shown that MPs are essential in through direct cell-cell contacts involving VCAM-1–VLA-4, orchestration of the muscle repair process (Grounds, 1987; ICAM-1–LFA-1, PECAM-1–PECAM-1 and CX3CL1- Lescaudron et al., 1999). CX3CR1 interactions. The decision of a cell to proliferate, differentiate or undergo apoptosis is an integrated response to its growth factors and adhesive environment (Schwartz and Ingber, 1994). At the population level, mpc growth depends on both cell-cycling activity and cell survival. Previous studies on mpc-supporting cues have focused on MP-released soluble growth factors (Robertson et al., 1993; Cantini and Carraro, 1995; Merly et al., 1999). Some particular growth factors, such as insulin growth factor I (IGF-I), in addition to being a potent myogenic differentiation factor (Tureckova et al., 2001), can both stimulate mpc proliferation in the presence of other soluble factors (Napier et al., 1999) and promote mpc survival (Lawlor and Rotwein, 2000). However, our previous (Chazaud et al., 2003b) and present studies indicate that MP-derived soluble factors globally stimulate mpc proliferation, as assessed by thymidine incorporation, whereas direct MP cell contacts confer protection against apoptosis to mpcs. Apoptotic cell death is a normal developmental event involving both proliferating myoblasts and postmitotic myofibres (Garcia-Martinez et al., 1993; Tidball et al., 1995; McClearn et al., 1995). As shown in our in vivo study, apoptosis of myogenic cells also occurs during regeneration of postnatal muscle. In this setting, TUNEL-positive myogenic cells disappear as MP infiltration proceeds. Obviously, this might reflect both MP phagocytosis of dead cells and the delivery of an MP pro-survival signal to living mpcs. In vitro, the protective effects of MPs were twofold stronger towards post-mitotic differentiating mpcs than towards cycling myoblasts. The physiological significance of this finding remains elusive. Furthermore, mpcs are at risk of undergoing apoptosis for different reasons during the proliferation and differentiation process. Fast-cycling myoblasts must face difficulties in maintaining adequate DNA repair that might constitute an intrinsic signal for apoptosis (Wang and Walsh, 1996). Then, as they withdraw from the cell cycle and begin to differentiate, mpcs are at particular risk of myoblast-fusion-associated apoptosis, induced by endoplasmic reticulum stress (Nakanishi et al., 2005). Finally, myotubes elongate through additional myoblast fusion and must progressively stabilise their structure by establishing close association with the extracellular matrix (ECM) (Huppertz et al., 2001). Myogenic cell adhesion to the Fig. 5. Functionality of candidate effectors at the mpc cell microenvironment seems to be crucial for their survival, as membrane; apoptosis assays. STS-treated mpcs were co-cultured demonstrated by increased muscle cell apoptosis associated with or without MPs in the presence or absence of antibodies with deficiencies in ECM-binding proteins such as 5 and directed against CX3CL1 and CX3CR1, VCAM-1 and VLA-4, 71 integrins, and ECM proteins such as laminins (Vachon ICAM-1 and LFA-1, or PECAM-1 (see bottom of figure). et al., 1996; Vachon et al., 1997; Miyagoe et al., 1997; Taverna (A,B) Apoptosis of mpcs was evaluated by annexin V labelling (A) et al., 1998; Montanaro et al., 1999). It is possible that MP- and DIOC-6 staining (B) after exclusion of CD14 cells. supportive cues help myotubes to achieve their adhesion- (C) Myoblast (white bars) and myotube (black bars) apoptosis was induced stabilisation safely. In line with this view, myotubes, evaluated by caspase-3 activity measurement. Results are expressed which poorly express Bcl-2 and are therefore more sensitive as percentage of apoptosis in STS-treated mpcs and are means ± s.e.m. of at least three experiments. than myoblasts to STS-induced apoptosis (Dominov et al., Journal of Cell Science Macrophages and myogenic cell survival 2503 Fig. 6. Apoptosis and muscle regeneration. Notexin-treated mouse muscle was labelled with a set of antibodies at different times after injury. (A) Example of apoptotic (red) and desmin + myogenic cells (green) at 3, 6 and 24 hours post-injury. MP infiltration was evaluated after F4/80 immunolabelling (blue curve) or CD11b immunolabelling (red curve) according to a 0-5 scale and the total number of apoptotic cells per field (black curve) was estimated. Among total apoptotic cells (white bars), apoptotic desmin-positive myogenic cells (black bars) was estimated at each time point. (B) Examples of immunolabellings of myogenic (desmin ) and macrophagic (CD11b ) cells for the anti- apoptotic molecular systems 3 days after injury. Blue: DAPI nuclei staining. Bars, 10 m. 1998), are endowed with stronger expression of the four MPs with mpcs showed increased Akt and ERK1/2 receptors involved in adhesion-induced pro-survival phosphorylation, increased Bcl-2 expression and decreased signalling. caspase-3 activity. VLA-4, but not LFA-1, PECAM-1 and CX3CR1, was We previously showed that, early after activation, mpcs previously reported to be expressed by mpcs and to increase secrete a set of chemoattractants to initiate recruitment of with myogenic differentiation (Rosen et al., 1992). The authors circulating monocytes into damaged muscle (Chazaud et al., evaluated VLA-4 as an ECM receptor (Rosen et al., 1992), 2003b). Once recruited, monocytes differentiate into MPs, although this integrin, like LFA-1, is also involved in cell-cell which are monocyte-derived MPs expressing VCAM-1, adhesion and signalling. All four receptors expressed by mpcs, ICAM-1, PECAM-1 and CX3CL1, as shown herein. The upon binding of their respective ligands VCAM-1, ICAM-1, newly recruited MPs release soluble factors that both amplify PECAM-1 and CX3CL1, were previously shown to mediate recruitment of MPs and stimulate mpc proliferation (Chazaud anti-apoptotic signalling in a variety of non-muscle cell types et al., 2003b). In addition, according to our DNA array, soluble (see Results section). VLA-4, PECAM-1 and CX3CR1 factors produced by MPs reinforce mpc expression of VLA-4, mediate activation of the PI 3-kinase/Akt survival pathway LFA-1, PECAM-1 and CX3CR1 by 20-80%. Thus, when MPs (Meucci et al., 2000; Gao et al., 2003; Ferrero et al., 2003; enter into contact with mpcs, both cell types appropriately Deiva et al., 2004) and, in addition, CX3CR1 activates ERK1/2 express anti-apoptotic ligands and counterreceptors. (Brand et al., 2002; Deiva et al., 2004). These signalling In conclusion, the present study highlights the complex pathways are both involved in mpc survival (Lawlor and network of intercellular signalling and communication Rotwein, 2000). Among many pro-survival effects, Akt involved in the organisation of the stromal support of phosphorylates BAD, causing its release from the complex it myogenesis. Our data indicating that inflammatory cells, i.e. forms with Bcl-2, allowing Bcl-2 to exert its anti-apoptotic macrophages, are beneficial for muscle regeneration are in activity freely (Song et al., 2005). Both Akt and ERK1/2 accordance with in vivo studies showing that blocking pathways lead to inhibition of caspase-3 (Allan et al., 2003; inflammation with anti-inflammatory drugs might be Song et al., 2005), the major effector of the last step of muscle deleterious for muscle regeneration and repair (Mishra et al., cell apoptosis (Tews, 2002). Consistently, co-cultures of 1995; Shen et al., 2005). Moreover, evidence that a set of Journal of Cell Science 2504 Journal of Cell Science 119 (12) adhesion molecules rescue mpcs from apoptosis might open the possibility of improving myoblast transfer therapy. A strong limitation of this therapeutic approach consists of early massive cell death of non-mechanical origin (Chazaud et al., 2003a), affecting >95% of transplanted mpcs (Skuk and Tremblay, 2000). Moreover, mpcs induced to proliferate actively ex vivo to obtain a huge number of cells for transplantation was shown to increase their susceptibility to undergo apoptosis upon deprivation of extrinsic supportive cues (Rehfeldt et al., 2004). It seems likely that the use of anti- apoptotic cells or molecules could limit massive transplanted cell death, thus allowing appropriated mpc proliferation, differentiation and striated muscle repair. Materials and Methods Cell cultures Unless indicated, culture media components were from Invitrogen (Gibco) and culture plastics from TPP AG (Trasadingen). Human mpcs were cultured from muscle samples as previously described (Chazaud et al., 2003b). Only cultures presenting over 95% CD56 cells (immunolabelling using anti-CD56 antibodies, diluted 1/20; Sanbio/Monosan) were used. Growing medium [HAM-F12 medium containing 15% fetal calf serum (FCS)] was used for culturing mpcs. To obtain myotubes, medium was replaced by HAM-F12 medium containing 5% FCS (differentiating medium) at time of subconfluence and cells were further cultured during 10 days (Lafuste et al., 2005). MPs were obtained from monocytes isolated from human blood as previously described (Chazaud et al., 2003b). Briefly, monocytes were seeded at 0.510 cell/ml in Teflon bags (AFC) in RPMI medium containing 15% human AB serum for 8 days. Cell treatments and co-cultures In each series of experiments, the number of mpcs remained constant whereas the number of MPs varied. Undifferentiated mpcs were seeded at 10,000 cells/cm . Differentiated myotubes were counted in order to seed the appropriate number of MPs, from 1:10 ratio. Co-cultures were incubated in growing or differentiating medium for 6 or 24 hours at 37°C. In some experiments, mpc apoptosis was first induced by staurosporine (STS) treatment (1 M for 6 hours). In some experiments, blocking antibodies were added in co-cultures of mpcs with MPs at saturating concentrations (calculated from IC50 or from previous studies): anti-CX3CL1 (3 g/ml, 51637.1 clone; R&D Systems) (Chazaud et al., 2003b), anti-CX3CR1 (15 g/ml, TP502; Torrey Pines Biolabs) (Chapman et al., 2000), anti-VCAM-1 (5 g/ml, 1G11 clone; Immunotech) (Minges Wols et al., 2002), anti-VLA-4 (5 g/ml, HP2/1 clone; Immunotech) (Hayashida et al., 2000), anti-LFA-1 (5 g/ml, TS1/22 clone; Endogen) (Hayashida et al., 2000), anti-ICAM-1 (5 g/ml, 84H10 clone; Fig. 7. Measurement of mpc apoptosis. (A) Example of flow Immunotech) (Winter et al., 2001), anti-PECAM-1 (5 g/ml, VM64 clone, cytometric analysis of mpc apoptosis in co-cultures of mpcs with Biodesign International). In other experiments, co-cultures were performed in the MPs. CD14 labelling is used to discriminate MPs from mpcs. The presence of hydrogen peroxide (0.2 mM; Sigma) (Anderson et al., 2002) or + – apoptotic mpc population is gated in red: annexin V CD14 cells cytochalasin D (1 g/ml, Sigma) (Elliott and Winn, 1986; Rubartelli et al., 1997). – – Controls included addition of whole IgGs from mouse and rabbit (3 g/ml; Vector and DIOC-6 CD14 cells. (B) Example of spontaneous (dotted Laboratories). lines) and STS-induced (continuous lines) apoptosis in mpc cultures. (C) Expression of CD14 by CD45 cells in co-cultures of mpcs with Measurement of mpc apoptosis by flow cytometry MPs. Trypsin was used to detach mpcs and detection of apoptotic cells was performed using annexin V plus CD14 labelling and DIOC-6 plus CD14 staining. CD14 labelling was used to exclude MPs detached by the trypsinisation procedure (Fig. 7A). Cells were resuspended in 100 l buffer (140 mM NaCl, 2.5 mM CaCl , 10 recovered after centrifugation at 4000 g for 10 minutes at 4°C. Protein concentration was determined using the BCA protein assay kit from Pierce. Aliquots mM HEPES pH 7.4) containing either 2 l of annexin V (Roche Diagnostics) or 70 nM of DIOC-6 (Molecular Probes) and 10 l of TRITC-conjugated anti-CD14 corresponding to 30 g of proteins were diluted in caspase-3 reaction buffer (1 M Hepes pH 7.4, 40 mM EDTA pH 8.0, 100 mM DTT, 25% sucrose) and incubated antibodies (RMO52; Immunotech) for 30 minutes. Cells were washed before analysis by flow cytometry on a FACSCalibur (BD Biosciences). Apoptosis of mpcs during 8 hours at 37°C in a microplate with caspase-3 substrate (Ac-DEVD-AFC fluorogenic substrate, Biomol Research Laboratories). Enzymatic activity was was significantly increased by STS treatment, reaching 30±13% of the cells (annexin V detection) and 44±13% of the cells (DIOC-6 detection) (P<0.01) (Fig. measured every 30 minutes with a fluorescence plate reader FL600 (Bio-Tek) and was expressed in arbitrary units. 7B). As the range of apoptotic mpcs was 19-60% (annexin V detection) and 30- 70% (DIOC-6 detection) of the cells, mpc apoptosis was expressed in percentage of apoptosis evaluated in STS-treated mpc cultures (without MPs). In co-cultures Adhesion of mpcs on MPs of MPs with untreated mpcs, CD14 expression was not affected (Fig. 7C); in co- Before being allowed to adhere on a confluent monolayer of MPs at various densities cultures of MPs with STS-treated mpcs, we observed no more than 4-5% of CD14 (5000 to 50,000 mpcs per well) and for various times (30 to 120 minutes), mpcs cells among CD45 cells (Fig. 7C), indicating that gating allowed exclusion of were labelled with 5-bromo-2-deoxyuridine (BrdU) for 72 hours. BrdU was then >95% of MPs. quantified using a colorimetric assay (Cell proliferation ELISA BrdU kit; R&D Systems). Measurement of caspase-3 activity Proteins from mpcs, MP cultures and mpc-MP co-cultures were extracted in lysis Adhesion of mpcs on ligand coats buffer (50 mM Hepes pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 40 mM EGTA pH Flat-bottomed 96-well sterile plates were coated with human recombinant VCAM- 8.0, 100 mM DTT, 2 g/ml leupeptin, 2 g/ml aprotinin, 1 g/ml pepstatin) and 1, CX3CL1, ICAM-1 or PECAM-1 (0.001 to 100 nM) (R&D Systems) in Journal of Cell Science Macrophages and myogenic cell survival 2505 phosphate-buffered saline (PBS). Non-specific binding sites were blocked with 1% In vivo immunolabellings bovine serum albumin for 30 minutes at room temperature. 30,000 mpcs per well Muscle cryosections were double labelled with either desmin antibodies (60 g/ml; were allowed to adhere for 2 hours at 37°C. Non-adherent cells were removed by Abcam) and anti-VLA-4 (15 g/ml, Chemicon International) or anti-LFA-1 (10 gentle PBS washes. Cells were fixed with acetone and methanol for 15 minutes and g/ml; Abcam) or anti-PECAM-1 (10 g/ml, Santa Cruz) or anti-CX3CR1 (10 stained with 0.5% Violet Crystal for 15 minutes. The number of adherent cells was g/ml; R&D Systems, using the MOM kit from Vector Laboratories) antibodies to evaluated by reading the OD at 540 nm. detect mpc expression. Slides were treated with anti-CD11b antibodies (10 g/ml; BD Biosciences) and anti-CX3CL1 (15 g/ml; Abcam) or anti-VCAM-1 (15 g/ml; R&D Systems) or anti-ICAM-1 (50 g/ml; Chemicon International) or anti- DNA array PECAM-1 (10 g/ml; Santa Cruz) antibodies to detect MP expression. To evaluate Total RNA was prepared from human mpcs and MPs using the RNeasy mini kit MP infiltration after injury, slides were treated with anti-CD11b as above or anti- (Qiagen). All further steps were performed according to the manufacturer’s F4/80 antibodies (20 g/ml; Abcam). Primary antibodies were detected with either instructions in the human cytokine array GA001 kit (R&D Systems). For mpc and cy3-labelled or FITC-labelled secondary antibodies (Jackson ImmunoResearch MP samples, 5 and 7 g of total RNA gave labelled cDNA of 600,000 and 800,000 Laboratories). Controls included incubation with whole IgGs from species of the cpm, respectively, which was deposited on membranes. Results were read using a primary antibody (Vector Laboratories). Slides were examined as described above. Phosphorimager (Amersham) after 72 hours exposure time. Analysis was performed using Image Quant software (Amersham), which allows background noise subtraction, correction for the variation of density for housekeeping genes and, Statistical analyses finally, for comparison of densitometric signals. Results were expressed in arbitrary Except DNA array, all experiments were performed using at least three different units. cultures or animals. The Student’s t-test and ANOVA analysis were used for statistical analyses. P<0.05 was considered significant. RT-PCR Total mpc or MP RNA (1.5 g) was reverse transcribed and amplified using This work was supported by the Association Française contre les OneStep RTPCR (Qiagen) and specific primers. For CX3CL1 [primers described in Myopathies and Fondation de France. We thank S. Lotersztajn for Lucas et al. (Lucas et al., 2001)], amplification was performed at 94, 64 and 72°C helpful discussions. for 30 seconds, 30 seconds and 1 minute, respectively, for 38 cycles. For CX3CR1 [primers described in Muehlhoefer et al. (Muehlhoefer et al., 2000)], amplification References was performed at 94, 55 and 72°C for 30 seconds, 30 seconds and 45 seconds, Allan, L. A., Morrice, N., Brady, S., Magee, G., Pathak, S. and Clarke, P. R. (2003). respectively. For VCAM-1 [primers described in Serradell et al. (Serradell et al., Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat. Cell 2002)], amplification was performed at 94, 53 and 72°C for 30 seconds, 30 seconds Biol. 5, 647-654. and 45 seconds, respectively, for 38 cycles. For 4 integrin (GenBank # Anderson, H. A., Englert, R., Gursel, I. and Shacter, E. (2002). Oxidative stress inhibits NM_000885), the sense primer used was 5-CGA ACC GAT GGC TCC TA-3 and the phagocytosis of apoptotic cells that have externalized phosphatidylserine. Cell the antisense primer was 5-AGT ATG CTG GCT CCG AAA AT-3, amplification Death Differ. 9, 616-625. was performed at 94, 55 and 72°C for 30 seconds, 30 seconds and 45 seconds, Bazan, J. F., Bacon, K. B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D. respectively, for 40 cycles. For ICAM-1 [primers described in Besch et al. (Besch R., Zlotnik, A. and Schall, T. J. (1997). A new class of membrane-bound chemokine et al., 2002)], amplification was performed at 94, 65 and 72°C for 30 seconds, 30 with a CX3C motif. Nature 385, 640-644. seconds and 45 seconds, respectively, for 50 cycles. For L integrin (GenBank # Besch, R., Giovannangeli, C., Kammerbauer, C. and Degitz, K. (2002). Specific BC008777), the sense primer used was 5-TTT GAG AAG AAC TGT GGG GAG inhibition of ICAM-1 expression mediated by gene targeting with Triplex-forming GAC-3 and the antisense primer was 5-GGT GGG CGA GAT GGA AGG T-3, oligonucleotides. J. Biol. Chem. 277, 32473-32479. both amplification was performed at 94, 60 and 72°C for 30 seconds, 45 seconds Bird, I. N., Taylor, V., Newton, J. P., Spragg, J. H., Simmons, D. L., Salmon, M. and and 2 minutes, respectively, for 40 cycles. Amplification products (10 l) were Buckley, C. D. (1999). Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration. J. Cell Sci. 112, 1989- subjected to electrophoresis on 2% agarose gel containing ethidium bromide for visualisation. Blasi, E., Back, T. C., Stull, S. W. and Varesio, L. (1987). Regulation of bone marrow cell survival in short-term cultures: a new macrophage function. Cell Immunol. 104, Immunoblotting 334-342. Total proteins from mpc and MP cultures, and co-cultures of mpcs with MPs, were Boehme, S. A., Lio, F. M., Maciejewski-Lenoir, D., Bacon, K. B. and Conlon, P. J. extracted as described in Davaille et al. (Davaille et al., 2002). Protein concentration (2000). The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia. was determined using the BCA protein assay kit. Aliquots corresponding to 15 g J. Immunol. 165, 397-403. of proteins were subjected to western blot. Anti-phosphorylated Akt (1/1000; Cell Borthwick, N. J., Akbar, A. A., Buckley, C., Pilling, D., Salmon, M., Jewell, A. P. and Signalling Technology), anti-phosphorylated ERK1/2 (1/1000; Promega), anti-Bcl- Yong, K. L. (2003). Transendothelial migration confers a survival advantage to 2 (1/500; Santa Cruz Biotechnology), anti-PECAM-1 (1/500, Dakocytomation) or activated T lymphocytes: role of LFA-1/ICAM-1 interactions. Clin. Exp. Immunol. 134, anti--actin (1/1000; Santa Cruz Biotechnology) antibodies were added overnight 246-252. and revealed using peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat Brand, S., Sakaguchi, T., Gu, X., Colgan, S. P. and Reinecker, H. C. (2002). antibodies (1/4000; Santa Cruz), which was detected using a chemiluminescence Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses kit (Amersham Biosciences). in intestinal epithelial cells. Gastroenterology 122, 166-177. Burgess, D. H., Svensson, M., Dandrea, T., Gronlund, K., Hammarquist, F., Orrenius, S. and Cotgreave, I. A. (1999). Human skeletal muscle cytosols are refractory to In vitro immunolabellings cytochrome c-dependent activation of type-II caspases and lack APAF-1. Cell Death Human cells cultured on coverslips were labelled with primary antibodies Differ. 6, 256-261. (same references as above) for 2 hours: anti-CX3CL1 (50 g/ml), anti-CX3CR1 Cantini, M. and Carraro, U. (1995). Macrophage-released factor stimulates selectively (15 g/ml), anti-VCAM-1 (15 g/ml), anti-VLA-4 (15 g/ml), anti-ICAM-1 myogenic cells in primary muscle culture. J. Neuropathol. Exp. Neurol. 54, 121-128. (15 g/ml), anti-LFA-1 (15 g/ml), anti-PECAM-1 (15 g/ml), revealed using Cantini, M., Massimino, M. L., Bruson, A., Catani, C., Dalla, L. L. and Carraro, U. biotinylated antibody (1/200), HRP-streptavidine (1/200) and DAB substrate (1994). Macrophages regulate proliferation and differentiation of satellite cells. kit for peroxidase (Vector Laboratories). Controls included incubation with Biochem. Biophys. Res. Commun. 202, 1688-1696. whole IgGs from the species of the primary antibody (50 g/ml; Vector Chapman, G. A., Moores, K., Harrison, D., Campbell, C. A., Stewart, B. R. and Laboratories). Strijbos, P. J. (2000). Fractalkine cleavage from neuronal membranes represents an acute event in the inflammatory response to excitotoxic brain damage. J. Neurosci. 20, In vivo toxic muscle injury RC87. Charbord, P. and Moore, K. (2005). Gene expression in stem cell-supporting stromal Notexin (10 l of 25 g/ml in PBS; Sigma) was injected into the tibialis anterior cell lines. Ann. N.Y. Acad. Sci. 1044, 159-167. of adult C57/B6 mice. At various times after injection, muscles were removed, snap Charbord, P., Remy-Martin, J. P., Tamayo, E., Bernard, G., Keating, A. and Peault, frozen in nitrogen-chilled isopentane (–160°C) and kept at –80°C until use. 7 m- B. (2000). Analysis of the microenvironment necessary for engraftment: role of the thick cryosections were treated for immunolabelling. vascular smooth muscle-like stromal cells. J. Hematother. Stem Cell Res. 9, 935-943. Chazaud, B., Hittinger, L., Sonnet, C., Champagne, S., Le Corvoisier, P., Benhaiem- In situ detection of apoptosis (2003a). Sigaux, N., Unterseeh, T., Su, J., Merlet, P., Rahmouni, A. et al. Muscle cryosections were incubated with rabbit polyclonal desmin antibodies (60 Endoventricular porcine autologous myoblast transplantation can be successfully g/ml; Abcam) and further treated to detect apoptotic nuclei (Apoptag Red; achieved with minor mechanical cell damage. Cardiovasc. Res. 58, 444-450. Qbiogen). Slides were examined under an Axioplan 2 Zeiss microscope (Carl Zeiss) Chazaud, B., Sonnet, C., Lafuste, P., Bassez, G., Rimaniol, A. C., Poron, F., Authier, and images were captured with an Orca ER digital camera (Hamamatsu Photonics F. J., Dreyfus, P. A. and Gherardi, R. K. (2003b). Satellite cells attract monocytes KK) using Simple PCI software (C-Imaging Compix). Apoptotic desmin and and use macrophages as a support to escape apoptosis and enhance muscle growth. J. desmin cells were counted in at least 20 randomly chosen fields within the injured Cell Biol. 163, 1133-1143. area (20 objective). Columbaro, M., Mattioli, E., Lattanzi, G., Rutigliano, C., Ognibene, A., Maraldi, N. Journal of Cell Science 2506 Journal of Cell Science 119 (12) M. and Squarzoni, S. (2001). Staurosporine treatment and serum starvation promote and Authier, F. J. (2005). ADAM12 and alpha9beta1 integrin are instrumental in the cleavage of emerin in cultured mouse myoblasts: involvement of a caspase- human myogenic cell differentiation. Mol. Biol. Cell 16, 861-870. dependent mechanism. FEBS Lett. 509, 423-429. Lapidot, T. and Petit, I. (2002). Current understanding of stem cell mobilization: the Davaille, J., Li, L., Mallat, A. and Lotersztajn, S. (2002). Sphingosine 1-phosphate roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal triggers both apoptotic and survival signals for human hepatic myofibroblasts. J. Biol. cells. Exp. Hematol. 30, 973-981. Chem. 277, 37323-37330. Laskin, D. L. and Laskin, J. D. (2001). Role of macrophages and inflammatory Deiva, K., Geeraerts, T., Salim, H., Leclerc, P., Hery, C., Hugel, B., Freyssinet, J. M. mediators in chemically induced toxicity. Toxicology 160, 111-118. and Tardieu, M. (2004). Fractalkine reduces N-methyl-d-aspartate-induced calcium Lawlor, M. A. and Rotwein, P. (2000). Insulin-like growth factor-mediated muscle cell flux and apoptosis in human neurons through extracellular signal-regulated kinase survival: central roles for Akt and cyclin-dependent kinase inhibitor p21. Mol. Cell. activation. Eur. J. Neurosci. 20, 3222-3232. Biol. 20, 8983-8995. Dominov, J. A., Dunn, J. J. and Miller, J. B. (1998). Bcl-2 expression identifies an early Lefaucheur, J. P. and Sebille, A. (1995). The cellular events of injured muscle stage of myogenesis and promotes clonal expansion of muscle cells. J. Cell Biol. 142, regeneration depend on the nature of the injury. Neuromuscul. Disord. 5, 501-509. 537-544. Lescaudron, L., Peltekian, E., Fontaine-Perus, J., Paulin, D., Zampieri, M., Garcia, Du, J., Wang, X., Miereles, C., Bailey, J. L., Debigare, R., Zheng, B., Price, S. R. and L. and Parrish, E. (1999). Blood borne macrophages are essential for the triggering Mitch, W. E. (2004). Activation of caspase-3 is an initial step triggering accelerated of muscle regeneration following muscle transplant. Neuromuscul. Disord. 9, 72-80. muscle proteolysis in catabolic conditions. J. Clin. Invest. 113, 115-123. Leu, S. T., Jacques, S. A., Wingerd, K. L., Hikita, S. T., Tolhurst, E. C., Pring, J. L., Elliott, J. A. and Winn, W. C., Jr (1986). Treatment of alveolar macrophages with Wiswell, D., Kinney, L., Goodman, N. L., Jackson, D. Y. et al. (2004). Integrin cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. alpha4beta1 function is required for cell survival in developing retina. Dev. Biol. 276, Infect. Immun. 51, 31-36. 416-430. Evans, P. C., Taylor, E. R. and Kilshaw, P. J. (2001). Signaling through CD31 protects Leussink, V. I., Zettl, U. K., Jander, S., Pepinsky, R. B., Lobb, R. R., Stoll, G., Toyka, endothelial cells from apoptosis. Transplantation 71, 457-460. K. V. and Gold, R. (2002). Blockade of signaling via the very late antigen (VLA-4) Ferrero, E., Belloni, D., Contini, P., Foglieni, C., Ferrero, M. E., Fabbri, M., Poggi, and its counterligand vascular cell adhesion molecule-1 (VCAM-1) causes increased A. and Zocchi, M. R. (2003). Transendothelial migration leads to protection from T cell apoptosis in experimental autoimmune neuritis. Acta Neuropathol. 103, 131-136. starvation-induced apoptosis in CD34+CD14+ circulating precursors: evidence for Lucas, A. D., Chadwick, N., Warren, B. F., Jewell, D. P., Gordon, S., Powrie, F. and PECAM-1 involvement through Akt/PKB activation. Blood 101, 186-193. Greaves, D. R. (2001). The transmembrane form of the CX3CL1 chemokine Fong, A. M., Robinson, L. A., Steeber, D. A., Tedder, T. F., Yoshie, O., Imai, T. and fractalkine is expressed predominantly by epithelial cells in vivo. Am. J. Pathol. 158, Patel, D. D. (1998). Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte 855-866. capture, firm adhesion, and activation under physiologic flow. J. Exp. Med. 188, 1413- Massimino, M. L., Rapizzi, E., Cantini, M., Libera, L. D., Mazzoleni, F., Arslan, P. 1419. and Carraro, U. (1997). ED2+ macrophages increase selectively myoblast Gao, C., Sun, W., Christofidou-Solomidou, M., Sawada, M., Newman, D. K., Bergom, proliferation in muscle cultures. Biochem. Biophys. Res. Commun. 235, 754-759. C., Albelda, S. M., Matsuyama, S. and Newman, P. J. (2003). PECAM-1 functions Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, as a specific and potent inhibitor of mitochondrial-dependent apoptosis. Blood 102, 493-495. 169-179. McArdle, A., Maglara, A., Appleton, P., Watson, A. J., Grierson, I. and Jackson, M. Garcia-Martinez, V., Macias, D., Ganan, Y., Garcia-Lobo, J. M., Francia, M. V., J. (1999). Apoptosis in multinucleated skeletal muscle myotubes. Lab. Invest. 79, 1069- Fernandez-Teran, M. A. and Hurle, J. M. (1993). Internucleosomal DNA 1076. fragmentation and programmed cell death (apoptosis) in the interdigital tissue of the McClearn, D., Medville, R. and Noden, D. (1995). Muscle cell death during the embryonic chick leg bud. J. Cell Sci. 106, 201-208. development of head and neck muscles in the chick embryo. Dev. Dyn. 202, 365-377. Geske, F. J., Monks, J., Lehman, L. and Fadok, V. A. (2002). The role of McLennan, I. S. (1996). Degenerating and regenerating skeletal muscles contain several the macrophage in apoptosis: hunter, gatherer, and regulator. Int. J. Hematol. 76, 16- subpopulations of macrophages with distinct spatial and temporal distributions. J. Anat. 26. 188, 17-28. Goda, S., Imai, T., Yoshie, O., Yoneda, O., Inoue, H., Nagano, Y., Okazaki, T., Imai, Merly, F., Lescaudron, L., Rouaud, T., Crossin, F. and Gardahaut, M. F. (1999). H., Bloom, E. T., Domae, N. et al. (2000). CX3C-chemokine, fractalkine-enhanced Macrophages enhance muscle satellite cell proliferation and delay their differentiation. adhesion of THP-1 cells to endothelial cells through integrin-dependent and Muscle Nerve 22, 724-732. -independent mechanisms. J. Immunol. 164, 4313-4320. Meucci, O., Fatatis, A., Simen, A. A. and Miller, R. J. (2000). Expression of CX3CR1 Gordon, S. (1995). The macrophage. BioEssays 17, 977-986. chemokine receptors on neurons and their role in neuronal survival. Proc. Natl. Acad. Gras, G., Chretien, F., Vallat-Decouvelaere, A. V., Le Pavec, G., Porcheray, F., Sci. USA 97, 8075-8080. Bossuet, C., Leone, C., Mialocq, P., Dereuddre-Bosquet, N., Clayette, P. et al. Meyer, D. M., Dustin, M. L. and Carron, C. P. (1995). Characterization of intercellular (2003). Regulated expression of sodium-dependent glutamate transporters and adhesion molecule-1 ectodomain (sICAM-1) as an inhibitor of lymphocyte function- synthetase: a neuroprotective role for activated microglia and macrophages in HIV associated molecule-1 interaction with ICAM-1. J. Immunol. 155, 3578-3584. infection? Brain Pathol. 13, 211-222. Mierke, C. T., Ballmaier, M., Werner, U., Manns, M. P., Welte, K. and Bischoff, S. Grounds, M. D. (1987). Phagocytosis of necrotic muscle in muscle isografts is influenced C. (2000). Human endothelial cells regulate survival and proliferation of human mast by the strain, age, and sex of host mice. J. Pathol. 153, 71-82. cells. J. Exp. Med. 192, 801-811. Hall, B. M., Fortney, J. E., Taylor, L., Wood, H., Wang, L., Adams, S., Davis, S. and Minges Wols, H. A., Underhill, G. H., Kansas, G. S. and Witte, P. L. (2002). The role Gibson, L. F. (2004). Stromal cells expressing elevated VCAM-1 enhance survival of of bone marrow-derived stromal cells in the maintenance of plasma cell longevity. J. B lineage tumor cells. Cancer Lett. 207, 229-239. Immunol. 169, 4213-4221. Hanspal, M. and Hanspal, J. S. (1994). The association of erythroblasts with Mishra, D. K., Friden, J., Schmitz, M. C. and Lieber, R. L. (1995). Anti-inflammatory macrophages promotes erythroid proliferation and maturation: a 30-kD heparin- medication after muscle injury. A treatment resulting in short-term improvement but binding protein is involved in this contact. Blood 84, 3494-3504. subsequent loss of muscle function. J. Bone Joint Surg. Am. 77, 1510-1519. Harrison, J. K., Jiang, Y., Chen, S., Xia, Y., Maciejewski, D., McNamara, R. K., Miyagoe, Y., Hanaoka, K., Nonaka, I., Hayasaka, M., Nabeshima, Y., Arahata, K., Streit, W. J., Salafranca, M. N., Adhikari, S., Thompson, D. A. et al. (1998). Role Nabeshima, Y. and Takeda, S. (1997). Laminin alpha2 chain-null mutant mice by for neuronally derived fractalkine in mediating interactions between neurons and targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient CX3CR1-expressing microglia. Proc. Natl. Acad. Sci. USA 95, 10896-10901. congenital muscular dystrophy. FEBS Lett. 415, 33-39. Hawke, T. J. and Garry, D. J. (2001). Myogenic satellite cells: physiology to molecular Mizuno, T., Kawanokuchi, J., Numata, K. and Suzumura, A. (2003). Production and biology. J. Appl. Physiol. 91, 534-551. neuroprotective functions of fractalkine in the central nervous system. Brain Res. 979, Hayashida, K., Shimaoka, Y., Ochi, T. and Lipsky, P. E. (2000). Rheumatoid arthritis 65-70. synovial stromal cells inhibit apoptosis and up-regulate Bcl-xL expression by B cells Montanaro, F., Lindenbaum, M. and Carbonetto, S. (1999). alpha-Dystroglycan is a in a CD49/CD29-CD106-dependent mechanism. J. Immunol. 164, 1110-1116. laminin receptor involved in extracellular matrix assembly on myotubes and muscle Huppertz, B., Tews, D. S. and Kaufmann, P. (2001). Apoptosis and syncytial fusion in cell viability. J. Cell Biol. 145, 1325-1340. human placental trophoblast and skeletal muscle. Int. Rev. Cytol. 205, 215-253. Muehlhoefer, A., Saubermann, L. J., Gu, X., Luedtke-Heckenkamp, K., Xavier, R., Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, Blumberg, R. S., Podolsky, D. K., MacDermott, R. P. and Reinecker, H. C. (2000). M., Takagi, S., Nomiyama, H., Schall, T. J. et al. (1997). Identification and molecular Fractalkine is an epithelial and endothelial cell-derived chemoattractant for characterization of fractalkine receptor CX3CR1, which mediates both leukocyte intraepithelial lymphocytes in the small intestinal mucosa. J. Immunol. 164, 3368- migration and adhesion. Cell 91, 521-530. 3376. Jejurikar, S. S. and Kuzon, W. M., Jr (2003). Satellite cell depletion in degenerative Muller-Sieburg, C. E. and Deryugina, E. (1995). The stromal cells’ guide to the stem skeletal muscle. Apoptosis 8, 573-578. cell universe. Stem Cells 13, 477-486. Koopman, G., Keehnen, R. M., Lindhout, E., Newman, W., Shimizu, Y., van Seventer, Nakanishi, K., Sudo, T. and Morishima, N. (2005). Endoplasmic reticulum stress G. A., de Groot, C. and Pals, S. T. (1994). Adhesion through the LFA-1 signaling transmitted by ATF6 mediates apoptosis during muscle development. J. Cell (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) Biol. 169, 555-560. pathways prevents apoptosis of germinal center B cells. J. Immunol. 152, 3760-3767. Napier, J. R., Thomas, M. F., Sharma, M., Hodgkinson, S. C. and Bass, J. J. (1999). Koseki, T., Inohara, N., Chen, S. and Nunez, G. (1998). ARC, an inhibitor of apoptosis Insulin-like growth factor-I protects myoblasts from apoptosis but requires other factors expressed in skeletal muscle and heart that interacts selectively with caspases. Proc. to stimulate proliferation. J. Endocrinol. 163, 63-68. Natl. Acad. Sci. USA 95, 5156-5160. Oostendorp, R. A., Reisbach, G., Spitzer, E., Thalmeier, K., Dienemann, H., Lafuste, P., Sonnet, C., Chazaud, B., Dreyfus, P. A., Gherardi, R. K., Wewer, U. M. Mergenthaler, H. G. and Dormer, P. (1995). VLA-4 and VCAM-1 are the principal Journal of Cell Science Macrophages and myogenic cell survival 2507 adhesion molecules involved in the interaction between blast colony-forming cells and Song, G., Ouyang, G. and Bao, S. (2005). The activation of Akt/PKB signaling pathway bone marrow stromal cells. Br. J. Haematol. 91, 275-284. and cell survival. J. Cell Mol. Med. 9, 59-71. Ostrovsky, O. and Bengal, E. (2003). The mitogen-activated protein kinase cascade Takeishi, T., Hirano, K., Kobayashi, T., Hasegawa, G., Hatakeyama, K. and Naito, promotes myoblast cell survival by stabilizing the cyclin-dependent kinase inhibitor, M. (1999). The role of Kupffer cells in liver regeneration. Arch. Histol. Cytol. 62, 413- p21WAF1 protein. J. Biol. Chem. 278, 21221-21231. 422. Pimorady-Esfahani, A., Grounds, M. D. and McMenamin, P. G. (1997). Macrophages Taverna, D., Disatnik, M. H., Rayburn, H., Bronson, R. T., Yang, J., Rando, T. A. and dendritic cells in normal and regenerating murine skeletal muscle. Muscle Nerve and Hynes, R. O. (1998). Dystrophic muscle in mice chimeric for expression of alpha5 20, 158-166. integrin. J. Cell Biol. 143, 849-859. Polazzi, E., Gianni, T. and Contestabile, A. (2001). Microglial cells protect cerebellar Tews, D. S. (2002). Apoptosis and muscle fibre loss in neuromuscular disorders. granule neurons from apoptosis: evidence for reciprocal signaling. Glia 36, 271-280. Neuromuscul. Disord. 12, 613-622. Rehfeldt, C., Renne, U., Wittstock, M., Mix, E. and Zettl, U. K. (2004). Long-term Tidball, J. G., Albrecht, D. E., Lokensgard, B. E. and Spencer, M. J. (1995). Apoptosis growth selection of mice changes the intrinsic susceptibility of myogenic cells to precedes necrosis of dystrophin-deficient muscle. J. Cell Sci. 108, 2197-2204. apoptosis. J. Muscle Res. Cell Motil. 25, 177-185. Tureckova, J., Wilson, E. M., Cappalonga, J. L. and Rotwein, P. (2001). Insulin-like Reuveny, M., Heller, H. and Bengal, E. (2004). RhoA controls myoblast survival by growth factor-mediated muscle differentiation: collaboration between inducing the phosphatidylinositol 3-kinase-Akt signaling pathway. FEBS Lett. 569, phosphatidylinositol 3-kinase-Akt-signaling pathways and myogenin. J. Biol. Chem. 129-134. 276, 39264-39270. Robertson, T. A., Maley, M. A., Grounds, M. D. and Papadimitriou, J. M. (1993). Umehara, H., Goda, S., Imai, T., Nagano, Y., Minami, Y., Tanaka, Y., Okazaki, T., The role of macrophages in skeletal muscle regeneration with particular reference to Bloom, E. T. and Domae, N. (2001). Fractalkine, a CX3C-chemokine, functions chemotaxis. Exp. Cell Res. 207, 321-331. predominantly as an adhesion molecule in monocytic cell line THP-1. Immunol. Cell Rose, D. M., Cardarelli, P. M., Cobb, R. R. and Ginsberg, M. H. (2000). Soluble Biol. 79, 298-302. VCAM-1 binding to alpha4 integrins is cell-type specific and activation dependent and Vachon, P. H., Loechel, F., Xu, H., Wewer, U. M. and Engvall, E. (1996). Merosin and is disrupted during apoptosis in T cells. Blood 95, 602-609. laminin in myogenesis; specific requirement for merosin in myotube stability and Rosen, G. D., Sanes, J. R., LaChance, R., Cunningham, J. M., Roman, J. and Dean, survival. J. Cell Biol. 134, 1483-1497. D. C. (1992). Roles for the integrin VLA-4 and its counter receptor VCAM-1 in Vachon, P. H., Xu, H., Liu, L., Loechel, F., Hayashi, Y., Arahata, K., Reed, J. C., myogenesis. Cell 69, 1107-1119. Wewer, U. M. and Engvall, E. (1997). Integrins (alpha7beta1) in muscle function and Rubartelli, A., Poggi, A. and Zocchi, M. R. (1997). The selective engulfment of survival. Disrupted expression in merosin-deficient congenital muscular dystrophy. J. apoptotic bodies by dendritic cells is mediated by the alpha(v)beta3 integrin and Clin. Invest. 100, 1870-1881. requires intracellular and extracellular calcium. Eur. J. Immunol. 27, 1893-1900. Wang, J. and Walsh, K. (1996). Resistance to apoptosis conferred by Cdk inhibitors Ruest, L. B., Khalyfa, A. and Wang, E. (2002). Development-dependent disappearance during myocyte differentiation. Science 273, 359-361. of caspase-3 in skeletal muscle is post-transcriptionally regulated. J. Cell Biochem. 86, Wang, J. and Lenardo, M. J. (1997). Essential lymphocyte function associated 1 (LFA- 21-28. 1): intercellular adhesion molecule interactions for T cell-mediated B cell apoptosis by Sadahira, Y. and Mori, M. (1999). Role of the macrophage in erythropoiesis. Pathol. Fas/APO-1/CD95. J. Exp. Med. 186, 1171-1176. Int. 49, 841-848. Wang, M. W., Consoli, U., Lane, C. M., Durett, A., Lauppe, M. J., Champlin, R., Schwartz, M. A. and Ingber, D. E. (1994). Integrating with integrins. Mol. Biol. Cell 5, Andreeff, M. and Deisseroth, A. B. (1998). Rescue from apoptosis in early (CD34- 389-393. selected) versus late (non-CD34-selected) human hematopoietic cells by very late Serradell, M., Diaz-Ricart, M., Cases, A., Zurbano, M. J., Lopez-Pedret, J., Arranz, antigen 4- and vascular cell adhesion molecule (VCAM) 1-dependent adhesion to bone O., Ordinas, A. and Escolar, G. (2002). Uremic medium causes expression, marrow stromal cells. Cell Growth Differ. 9, 105-112. redistribution and shedding of adhesion molecules in cultured endothelial cells. Winter, S. S., Sweatman, J. J., Lawrence, M. B., Rhoades, T. H., Hart, A. L. and Haematologica 87, 1053-1061. Larson, R. S. (2001). Enhanced T-lineage acute lymphoblastic leukaemia cell survival Shen, W., Li, Y., Tang, Y., Cummins, J. and Huard, J. (2005). NS-398, a on bone marrow stroma requires involvement of LFA-1 and ICAM-1. Br. J. Haematol. cyclooxygenase-2-specific inhibitor, delays skeletal muscle healing by decreasing 115, 862-871. regeneration and promoting fibrosis. Am. J. Pathol. 167, 1105-1117. Zen, K., Masuda, J. and Ogata, J. (1996). Monocyte-derived macrophages prime Skuk, D. and Tremblay, J. P. (2000). Progress in myoblast transplantation: a potential peripheral T cells to undergo apoptosis by cell-cell contact via ICAM-1/LFA-1- treatment of dystrophies. Microsc. Res. Tech. 48, 213-222. dependent mechanism. Immunobiology 195, 323-333. 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