Synergistic cytotoxic effect of interferon-gamma and tumor necrosis factor-alpha on cultured human muscle cells.
OBJECTIVE
To investigate the effects of human interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination, on the viability of human muscle cells in culture.


METHODS
Cultures of human muscle cells were treated with various concentrations of recombinant IFN-gamma and TNF-alpha alone and in combination, and the cytotoxic effects of the cytokines on muscle cells were assessed by measuring lactic dehydrogenase (LDH) release in supernatants and by observation of the cells for morphologic changes under phase microscopy.


RESULTS
Exposure of muscle cells to 100 U/ml of either IFN-gamma or TNF-alpha for 9 days caused no cytotoxic effects, as assessed by LDH release in supernatants of muscle cell cultures and by microscopic observation of the cell cultures. However, when IFN-gamma and TNF-alpha were added together in the muscle cell cultures, they caused significant cytotoxic effects. Thus, in combination, IFN-gamma and TNF-alpha at 100 U/ml each caused significant release of LDH (3rd day 9%, 4th day 28.5%, 7th day 55.5%, 9th day 74%) in the supernatants of treated cultures compared to controls. Moreover, inspection by phase microscopy showed clear damage of muscle cells; from Days 3 to 4 progressive vacuolation, detachment of cells, and finally disintegration of the muscle cells by the 8th to 10th day was observed. The synergistic cytotoxic effect of IFN-gamma and TNF-alpha occurred at concentrations as low as 1 U/ml and 10 U/ml, respectively.


CONCLUSIONS
Our study demonstrates for the first time a direct synergistic cytotoxic effect of IFN-gamma and TNF-alpha on human muscle cells in culture. Given that T cells and macrophages are prominent in the chronic inflammatory cell infiltrates of the affected muscles in patients with myositis, our findings suggest that IFN-gamma and TNF-alpha may play an important role in the pathogenesis of muscle destruction of this disorder.
Rheumatology Section, Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, IN 46202, USA.
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Kalovidouris
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Plotkin
D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D004357:Drug Synergism; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D024510:Muscle Development; D009132:Muscles / Q000166:cytology / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016898:Interferon-alpha; D014409:Tumor Necrosis Factor-alpha; D007770:L-Lactate Dehydrogenase