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Skeletal muscle interleukin 15 promotes CD8(+) T-cell function and autoimmune myositis.

Huang PL, Hou MS, Wang SW, Chang CL, Liou YH, Liao NS - Skelet Muscle (2015)

Bottom Line: Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ.On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8(+) T cells.These findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan ; Institute of Molecular Biology, Academia Sinica, Taipei, 11529 Taiwan.

ABSTRACT

Background: Interleukin 15 (IL-15) is thought to be abundant in the skeletal muscle under steady state conditions based on RNA expression; however, the IL-15 RNA level may not reflect the protein level due to post-transcriptional regulation. Although exogenous protein treatment and overexpression studies indicated IL-15 functions in the skeletal muscle, how the skeletal muscle cell uses IL-15 remains unclear. In myositis patients, IL-15 protein is up-regulated in the skeletal muscle. Given the supporting role of IL-15 in CD8(+) T-cell survival and activation and the pathogenic role of cytotoxic CD8(+) T cells in polymyositis and inclusion-body myositis, we hypothesize that IL-15 produced by the inflamed skeletal muscle promotes myositis via CD8(+) T cells.

Methods: Expression of IL-15 and IL-15 receptors at the protein level by skeletal muscle cells were examined under steady state and cytokine stimulation conditions. The functions of IL-15 in the skeletal muscle were investigated using Il15 knockout (Il15 (-/-) ) mice. The immune regulatory role of skeletal muscle IL-15 was determined by co-culturing cytokine-stimulated muscle cells and memory-like CD8(+) T cells in vitro and by inducing autoimmune myositis in skeletal-muscle-specific Il15 (-/-) mice.

Results: We found that the IL-15 protein was not expressed by skeletal muscle cells under steady state condition but induced by tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) stimulation and expressed as IL-15/IL-15 receptor alpha (IL-15Rα) complex. Skeletal muscle cells expressed a scanty amount of IL-15 receptor beta (IL-15Rβ) under either conditions and only responded to a high concentration of IL-15 hyperagonist, but not IL-15. Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ. On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8(+) T cells. Genetic ablation of Il15 in skeletal muscle cells greatly ameliorated autoimmune myositis in mice.

Conclusions: These findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.

No MeSH data available.


Related in: MedlinePlus

IL-15 is presented on the surface of skeletal muscle cells. a Expression of IL-15 and IL-15Rα on C2C12 myoblasts. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without cytokine (Control) for 24 h and then stained with anti-IL-15 and IL-15Rα antibodies for flow cytometry analysis. “Unstained” indicates cells without antibody staining. Data are representative of three independent experiments with similar results. b Quantification of IL-15 bound by cell surface IL-15Rα on C2C12 myotubes. C2C12 myotubes were first treated with TNF-α and IFN-γ for 24 h, incubated with acid glycine buffer to dissociate IL-15 from cell surface IL-15Rα, and then re-treated with exogenous IL-15. Cell lysates from each step were collected and the amount of IL-15/ IL-15Rα complex was measured by ELISA. Data are mean ± SEM of triplicates and representative of two independent experiments with similar results. ***p < 0.001
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Fig2: IL-15 is presented on the surface of skeletal muscle cells. a Expression of IL-15 and IL-15Rα on C2C12 myoblasts. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without cytokine (Control) for 24 h and then stained with anti-IL-15 and IL-15Rα antibodies for flow cytometry analysis. “Unstained” indicates cells without antibody staining. Data are representative of three independent experiments with similar results. b Quantification of IL-15 bound by cell surface IL-15Rα on C2C12 myotubes. C2C12 myotubes were first treated with TNF-α and IFN-γ for 24 h, incubated with acid glycine buffer to dissociate IL-15 from cell surface IL-15Rα, and then re-treated with exogenous IL-15. Cell lysates from each step were collected and the amount of IL-15/ IL-15Rα complex was measured by ELISA. Data are mean ± SEM of triplicates and representative of two independent experiments with similar results. ***p < 0.001

Mentions: As the majority of the cytokine-induced IL-15/IL-15Rα complex was in the lysates of the myoblast and myotube (Fig. 1d, e), we next examined whether the complex is present on the cell surface. We found that TNF-α and IFN-γ induced expression of IL-15 and IL-15Rα on the surface of C2C12 myoblasts as detected by flow cytometry (Fig. 2a). For the cytokine-treated C2C12 myotubes, dissociation of IL-15 from IL-15Rα on the cell surface by washing cells with acidic glycine buffer resulted in an 80 % reduction of IL-15/IL-15Rα in the cell lysate (Fig. 2b), indicating that 80 % of IL-15 was presented by IL-15Rα on the cell surface. This reduction was not due to alteration of IL-15Rα by the acid treatment, as the level of IL-15/IL-15Rα resumed following the addition of exogenous IL-15 (Fig. 2b). These results indicated that the majority of the cytokine-induced IL-15 was present on the surface of skeletal muscle cells as IL-15/IL-15Rα complex.Fig. 2


Skeletal muscle interleukin 15 promotes CD8(+) T-cell function and autoimmune myositis.

Huang PL, Hou MS, Wang SW, Chang CL, Liou YH, Liao NS - Skelet Muscle (2015)

IL-15 is presented on the surface of skeletal muscle cells. a Expression of IL-15 and IL-15Rα on C2C12 myoblasts. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without cytokine (Control) for 24 h and then stained with anti-IL-15 and IL-15Rα antibodies for flow cytometry analysis. “Unstained” indicates cells without antibody staining. Data are representative of three independent experiments with similar results. b Quantification of IL-15 bound by cell surface IL-15Rα on C2C12 myotubes. C2C12 myotubes were first treated with TNF-α and IFN-γ for 24 h, incubated with acid glycine buffer to dissociate IL-15 from cell surface IL-15Rα, and then re-treated with exogenous IL-15. Cell lysates from each step were collected and the amount of IL-15/ IL-15Rα complex was measured by ELISA. Data are mean ± SEM of triplicates and representative of two independent experiments with similar results. ***p < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4584479&req=5

Fig2: IL-15 is presented on the surface of skeletal muscle cells. a Expression of IL-15 and IL-15Rα on C2C12 myoblasts. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without cytokine (Control) for 24 h and then stained with anti-IL-15 and IL-15Rα antibodies for flow cytometry analysis. “Unstained” indicates cells without antibody staining. Data are representative of three independent experiments with similar results. b Quantification of IL-15 bound by cell surface IL-15Rα on C2C12 myotubes. C2C12 myotubes were first treated with TNF-α and IFN-γ for 24 h, incubated with acid glycine buffer to dissociate IL-15 from cell surface IL-15Rα, and then re-treated with exogenous IL-15. Cell lysates from each step were collected and the amount of IL-15/ IL-15Rα complex was measured by ELISA. Data are mean ± SEM of triplicates and representative of two independent experiments with similar results. ***p < 0.001
Mentions: As the majority of the cytokine-induced IL-15/IL-15Rα complex was in the lysates of the myoblast and myotube (Fig. 1d, e), we next examined whether the complex is present on the cell surface. We found that TNF-α and IFN-γ induced expression of IL-15 and IL-15Rα on the surface of C2C12 myoblasts as detected by flow cytometry (Fig. 2a). For the cytokine-treated C2C12 myotubes, dissociation of IL-15 from IL-15Rα on the cell surface by washing cells with acidic glycine buffer resulted in an 80 % reduction of IL-15/IL-15Rα in the cell lysate (Fig. 2b), indicating that 80 % of IL-15 was presented by IL-15Rα on the cell surface. This reduction was not due to alteration of IL-15Rα by the acid treatment, as the level of IL-15/IL-15Rα resumed following the addition of exogenous IL-15 (Fig. 2b). These results indicated that the majority of the cytokine-induced IL-15 was present on the surface of skeletal muscle cells as IL-15/IL-15Rα complex.Fig. 2

Bottom Line: Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ.On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8(+) T cells.These findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan ; Institute of Molecular Biology, Academia Sinica, Taipei, 11529 Taiwan.

ABSTRACT

Background: Interleukin 15 (IL-15) is thought to be abundant in the skeletal muscle under steady state conditions based on RNA expression; however, the IL-15 RNA level may not reflect the protein level due to post-transcriptional regulation. Although exogenous protein treatment and overexpression studies indicated IL-15 functions in the skeletal muscle, how the skeletal muscle cell uses IL-15 remains unclear. In myositis patients, IL-15 protein is up-regulated in the skeletal muscle. Given the supporting role of IL-15 in CD8(+) T-cell survival and activation and the pathogenic role of cytotoxic CD8(+) T cells in polymyositis and inclusion-body myositis, we hypothesize that IL-15 produced by the inflamed skeletal muscle promotes myositis via CD8(+) T cells.

Methods: Expression of IL-15 and IL-15 receptors at the protein level by skeletal muscle cells were examined under steady state and cytokine stimulation conditions. The functions of IL-15 in the skeletal muscle were investigated using Il15 knockout (Il15 (-/-) ) mice. The immune regulatory role of skeletal muscle IL-15 was determined by co-culturing cytokine-stimulated muscle cells and memory-like CD8(+) T cells in vitro and by inducing autoimmune myositis in skeletal-muscle-specific Il15 (-/-) mice.

Results: We found that the IL-15 protein was not expressed by skeletal muscle cells under steady state condition but induced by tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) stimulation and expressed as IL-15/IL-15 receptor alpha (IL-15Rα) complex. Skeletal muscle cells expressed a scanty amount of IL-15 receptor beta (IL-15Rβ) under either conditions and only responded to a high concentration of IL-15 hyperagonist, but not IL-15. Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ. On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8(+) T cells. Genetic ablation of Il15 in skeletal muscle cells greatly ameliorated autoimmune myositis in mice.

Conclusions: These findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.

No MeSH data available.


Related in: MedlinePlus