Injured skeletal muscle: The effect of neutrophil-derived superoxide production

Neutrophils are inflammatory cells known to accumulate within skeletal muscle in the hours following both injurious and non-injurious muscle activity. Elevated reactive oxygen species (ROS) in skeletal muscle following muscle contractions have been implicated to be generated by neutrophils. Evidence supporting the concept that neutrophils located within muscle are activated to produce ROS following muscle activity is lacking. Therefore, the purpose of this study was to test if skeletal myotubes exposed to injury or mechanical loading causes the release of factors that increase neutrophil superoxide (O₂·−) production. Human skeletal myotubes, were either scrape-injured or exposed to 0, 5, 10, or 30% strain (0.25 Hz; 2 hours). The conditioned medium was then collected 5 hours after the initiation of the experimental protocol. Percent lactate dehydrogenase (LDH) release from scraped or strained myotubes was used as a measure of injury. Neutrophils isolated from human blood were cultured with conditioned medium and assayed for their ability to produce O₂·− via the cytochrome c assay. The influence of the conditioned medium on neutrophil-derived O₂·− production was determined by measuring its ability to induce either a priming effect or direct activating effect. Significant LDI-I release from the scrape-injured (44.2% ± 4.9; mean ± SE) and the 30% strained (17.5% ± 2.9) myotubes (P ≤ 0.05) was observed. A significant priming effect was observed for conditioned medium obtained from scrape-injured myotubes and from 10% and 30% strained myotubes relative to control. Conditioned medium obtained from 5% strained myotubes did not result in a priming effect relative to conditioned medium obtained from control. A significant direct activating effect was observed for conditioned medium obtained from scrape-injured myotubes but not for conditioned medium from myotubes exposed to 30% strain. In conclusion, myotubes exposed to cyclic strain released one or more factors that primed neutrophils for O₂·− production in a manner that was dependent on a threshold of strain (>5%) and independent of myotube injury. In addition, the ability of myotubes to release factor(s) that directly activate neutrophils to produce O₂·− appears to be related to the nature of injury and/or magnitude of injury.