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First published online 23 May 2006
doi: 10.1242/jcs.02988

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

C. Sonnet^*, P. Lafuste^*, L. Arnold, M. Brigitte, F. Poron, F. Authier, F. Chrétien, R. K. Gherardi and B. 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

View larger version (31K): [in a new window]   Fig. 1. Inhibition of both spontaneous and induced mpc apoptosis by MPs. (A,B) untreated (A) and STS-treated (B) mpcs were co-cultured with MPs at various ratios and mpc apoptosis was evaluated by annexin V labelling (white bars) and DIOC-6 staining (black bars) after exclusion of CD14+ cells. In A, all mpc:MP ratios used were statistically different from mpcs (1:0 ratio) (P0.04); in B, all mpc:MP ratios used, except the 1:0.5 ratio, were statistically different from mpcs (1:0 ratio) (P0.07). (C,D) STS-treated myoblasts (C, Mb) and myotubes (D, MT) were co-cultured with or without MP (1:2 mpc:MP ratio). Apoptosis was evaluated by caspase-3 activity measurement. Results are means ± s.e.m. of at least three experiments.

View larger version (20K): [in a new window]   Fig. 2. Induction of pro-survival signals in co-cultures of mpcs with MPs. (A) Examples of immunoblots of Bcl-2, phosphorylated Akt (Akt-P) and phosphorylated ERK1/2 (ERK1/2-P) in myoblasts (Mb), myotubes (MT), MP cultures and co-cultures. Detection of ß-actin was used to check the protein amount deposited in each well. (B) Apoptosis of mpcs was detected with annexin V (white bars) and DIOC-6 (black bars) in co-cultures of mpcs with MPs in the presence of H2O2 at low concentration (0.2 mM) or cytochalasin D (1 µg/ml). Results are means ± s.e.m. of three experiments.

View larger version (38K): [in a new window]   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, ß2 integrins, and PECAM-1 mRNA in mpcs. ß2M is beta2microglobulin. (B) Immunolabelling of CX3CL1, VCAM-1, ICAM-1 and PECAM-1 on MPs (left panel) and of CX3CR1, VLA-4 and LFA-1 on mpcs (right panel), revealed by DAB substrate kit for peroxidase. Magnification, 300x. PECAM-1 expression in mpcs was assessed by immunoblotting. MT, myotube; Mb, myoblast.

View larger version (21K): [in a new window]   Fig. 4. Functionality of candidate effectors at the mpc cell membrane; adhesion assays. (A,B) Adhesion of mpcs on a MP monolayer according to incubation time (A) and mpc concentration (B). Adhesion of mpcs on VCAM-1 (C), CX3CL1 (D), ICAM-1 (E) and PECAM-1 (F) coats. Results are means ± s.e.m. of three experiments.

View larger version (16K): [in a new window]   Fig. 5. Functionality of candidate effectors at the mpc cell membrane; apoptosis assays. STS-treated mpcs were co-cultured with or without MPs in the presence or absence of antibodies directed against CX3CL1 and CX3CR1, VCAM-1 and VLA-4, ICAM-1 and LFA-1, or PECAM-1 (see bottom of figure). (A,B) Apoptosis of mpcs was evaluated by annexin V labelling (A) and DIOC-6 staining (B) after exclusion of CD14+ cells. (C) Myoblast (white bars) and myotube (black bars) apoptosis was evaluated by caspase-3 activity measurement. Results are expressed as percentage of apoptosis in STS-treated mpcs and are means ± s.e.m. of at least three experiments.

View larger version (81K): [in a new window]   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 µm.

View larger version (42K): [in a new window]   Fig. 7. Measurement of mpc apoptosis. (A) Example of flow cytometric analysis of mpc apoptosis in co-cultures of mpcs with MPs. CD14 labelling is used to discriminate MPs from mpcs. The apoptotic mpc population is gated in red: annexin V+ CD14- cells and DIOC-6- CD14- cells. (B) Example of spontaneous (dotted lines) and STS-induced (continuous lines) apoptosis in mpc cultures. (C) Expression of CD14 by CD45 cells in co-cultures of mpcs with MPs.