| Skeletal muscle injury results in neutrophil accumulation; however the identity of muscle cell-derived chemoattractants promoting neutrophil entry into muscle is unknown. We tested the hypotheses that: (1) the degree of neutrophil chemotaxis induced by mechanical strain is dependent on the magnitude of muscle cell (myotube) injury and(2) neutrophil chemoattractants released from mechanically strained myotubes are proteins. We further aimed to identify myotube-derived cytokines and reveal if one or more cytokines sensitive to mechanical strain contribute to the observed neutrophil chemotaxis. Human myotubes grown in culture were exposed to mechanical strain regimens (ST) of 0, -10, -30, -50 or - 70 KPa of pressure for 30 min. Protocols of ST 30, 50 and 70 were found to be injurious compared to ST 10. Additionally, each of the higher strain regimens was more injurious than the previous (ST 70 > ST 50 > ST 30). Conditioned media from ST 30, 50 and 70 promoted chemotaxis in human blood neutrophils in a manner that was dependent on the degree of injury (ST 70 > ST 50 > ST 30). Boiled conditioned media was not chemoattractant for neutrophils indicating that the nature of the chemoattractants was likely proteinaceous. The majority of cytokines screened with a multiplex kit (18 out of the 22) were detected constitutively. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 concentrations in conditioned media increased in a manner similar to the chemotactic response. Concentrations of IL-8 in conditioned media were detected below levels that induced neutrophil chemotaxis. Neutralization of IL-8; however, completely inhibited the chemotactic response for ST 30 and ST 50 and reduced the chemotactic response in ST 70 conditioned media by 40%. These data suggest that IL-8 is functioning in synergy with another factor to promote chemotaxis. Neutralization of MCP-1 did not reduce chemotaxis after ST 70. This study enhances our understanding of the immunobiology of skeletal muscle by revealing that myotube-derived IL-8 promotes neutrophil chemotaxis after injurious mechanical strain. |
