Differential expression of the IL-1 system components during in vitro myogenesis: Implication of IL-1β in induction of myogenic cell apoptosis

Abstract

We evaluated the expression of IL-1 system by normal human myogenic cells during in vitro myogenesis and the effect of exogenous IL-1b. Expression of IL-1a and b, IL-1 receptor antagonist (IL-1Ra), IL-1RI and II, IL-1R accessory protein (IL1RAcP) and IL-1b converting enzyme (ICE) was studied by immunocytochemistry, immunoblotting, ELISA and RT ± PCR. Cell proliferation was evaluated by [ 3 H]thymidine incorporation, cell fusion by flow cytometry and cell death by in situ end-labelling. Human normal myogenic cells constitutively produced IL-1b and ICE, with a maximum expression at time of cell fusion. IL-1Rs and IL-1RAcP expression reached a peak at time of commitment to fusion. Myogenic cells produced small amounts of IL-1Ra at latest stages of culture, and only the intracellular isoform. Exposure of cultures to exogenous IL-1b (1 ± 5 ng/ml) induced myogenic cell apoptosis, without effect on cell proliferation or fusion. IL1b-induced cell death was associated with morphological changes including spreading appearance of cells and alteration of cell alignment. We conclude that (1) human myogenic cells constitutively produce IL-1b; (2) IL-1 system components are differentially expressed during in vitro myogenesis; (3) IL-1 system participates to the coordinated regulation of cell density during normal myogenesis, which could serve to control the muscle mass in vivo.

Myeloid cell‐derived tumor necrosis factor‐alpha promotes sarcopenia and regulates muscle cell fusion with aging muscle fibers

“…There was no difference in either CD68 mRNA expression or the number of CD68+ macrophages in old wild‐type and TNF ‐ α ‐null mice (Figure 5g,h). We also tested whether TNF‐α ablation affected the expression of other inflammatory cytokines that can affect myogenesis (Authier et al, 1999; Lieskovska, Guo, & Derman, 2003). Our qPCR results showed that the expression of IFNγ, IL‐6, and IL‐1β did not differ significantly in 24‐month‐old, TNF ‐ α‐ null muscles compared to age‐matched wild‐type muscles (Figure 5i).…”

Increased apoptosis, huntingtin inclusions and altered differentiation in muscle cell cultures from Huntington's disease subjects

“…To check the ability of HD myoblasts to fuse and differentiate into multinucleated myotubes, we evaluated the fusion index calculated as the ratio of nuclei in myotubes to the total number of nuclei, the mean number of nuclei per myotube and the number of myotubes according to the number of nuclei per cell (2-4, 5-9, 49 nuclei). 37 At 7 days in the differentiating medium, we analyzed 10 fields from each of four separate cell cultures from each HD and control subject.…”

ADAM12 and α9β1Integrin Are Instrumental in Human Myogenic Cell Differentiation

“…Human mpc were cultured from muscle samples as described previously, in Ham's-F12 medium containing 15% fetal calf serum (FCS) (growth medium, GM) and antibiotics (Authier et al, 1999). Human mpc seeded at 5000 cells/ cm 2 undergo proliferation and at day 4 of culture (nearly confluence), the medium was replaced with Ham's-F12 medium containing 5% FCS (differentiation medium, DM) to induce myogenic differentiation characterized by mpc fusion into myotubes (Baroffio et al, 1993).…”

“…By using this procedure, mpc undergo standardized myogenic differentiation, including proliferation phase up to day 4, and then withdrawal of cell cycle to enter differentiation and formation of small myotubes (day 7), and growth of preformed myotubes through accretion of additional mpc (day 14). Only cultures presenting Ͼ95% cells expressing CD56/neural cell adhesion molecule (NCAM), a marker of myogenic cells (De Rossi et al, 2000) were used (clone 123C3, 1:20; Sanbio/ Monosan, Uden, The Netherlands) (Chazaud et al, 2003).mpc Growth and Differentiation mpc growth curves are established by counting the cells with trypan blue dye exclusion (Authier et al, 1999). Myogenic differentiation is assessed by expression of myogenin as recommended previously (Fujio et al, 1999).…”

“…Cultures were evaluated at the following stages: 1) proliferation stage (day 4 of culture, GM); 2) early differentiation stage (day 7 of culture, 3 d in DM); and 3) late differentiation stage (day 14 of culture, 10 d in DM). Experiments were run for each point at least on cultures from three individuals at passage 2.The fusion index that helps assessment of ability of cells to fuse into multinucleated cells was calculated as the ratio of nuclei in myotubes to the total number of nuclei (Hirsch et al, 1998;Authier et al, 1999). To evaluate separately both formation of small nascent myotubes (2-4 nuclei) and growth of preformed myotubes, we performed nuclear number assays as described previously (Horsley et al, 2001).…”