Null mutation of myeloperoxidase in mice prevents mechanical activation of neutrophil lysis of muscle cell membranes in vitro and in vivo

Abstract

Membrane lysis is a common and early defect in muscles experiencing acute injuries or inflammation. Although increased mechanical loading of muscles can induce inflammation and membrane lysis, whether mechanical loads applied to muscle can promote the activation and cytolytic capacity of inflammatory cells and thereby increase muscle damage is unknown. We tested whether mechanical loads applied to mouse muscle cells in vitro can increase membrane lysis, and whether neutrophil-mediated lysis of muscle cells is promoted by mechanical loads applied in vitro and in vivo. Cyclic loads applied to muscle cells for 24 h in vitro produced little muscle cell lysis. Similarly, the addition of neutrophils to muscle cell cultures in the presence of superoxide dismutase (SOD) produced little muscle cell lysis. However, when cyclic mechanical loads were applied to neutrophil-muscle co-cultures in the presence of SOD, there was a synergistic effect on muscle cell lysis, suggesting that mechanical loading activates neutrophil cytotoxicity. However, application of mechanical loads to co-cultures of muscle cells and neutrophils that are null mutants for myeloperoxidase (MPO) showed no mechanical activation of neutrophil cytotoxicity. This indicates that loading promotes neutrophil cytotoxicity via MPO. Activity assays confirmed that mechanical loading of neutrophil-muscle co-cultures significantly increased MPO activity. We further tested whether muscle membrane lysis in vivo was mediated by neutrophils when muscle was subjected to modified loading by using a mouse model of muscle reloading following a period of unloading. We observed that MPO −/− soleus muscles showed a significant 52% reduction in membrane lysis compared to wild-type mice, although the mutation did not decrease inflammatory cell extravasation. Together, these in vitro and in vivo findings show that mechanical loading activates neutrophil-mediated lysis of muscle cells through an MPO-dependent pathway.

Regulation and phylogeny of skeletal muscle regeneration

“…Their action on the necrotic tissue relies on proteolysis, oxidation and phagocytosis. Muscle-specific inhibition of neutrophil/monocyte activation results in a delay in regeneration upon acute injury (Nguyen et al, 2005).…”

Regulatory interactions between muscle and the immune system during muscle regeneration

“…CD206 is a mannose receptor that binds and internalizes sugar moieties on molecules present at high levels in inflamed tissue. In the context of muscle damage and regeneration, MPO is an important ligand for CD206 (101) because it serves a prominent role in muscle membrane lysis that is caused by neutrophils (79), and its ligation and internalization by M2 macrophages would reduce cytotoxicity. CD206 expression by M2 macrophages is promoted by anti-inflammatory cytokines, and its binding increases the expression of anti-inflammatory cytokines, leading to positive feedback that can enable M2 macrophages to more rapidly deactivate Th1 cells that are capable of free radical-mediated damage of muscle cells.…”

The Role of Neutrophils in Rheumatoid Arthritis – Experiments In Vitro: A Change of Conception?

“…Loading alone caused only a 1.7% lysis of muscle cells, while coculturing with neutrophils in the absence of loading resulted in only 3.5% lysis. However, loading in the presence of neutrophils resulted in 12.6% lysis of muscle cells under otherwise identical culture conditions in the presence of SOD (Nguyen et al, 2005). These observations provide a direct link between changes in mechanical loads applied to tissue, and an increase in damage that is induced by inflammatory cells.…”

“…Similarly, administration of catalase decreases hydrogen peroxide concentration and reduces tissue damage during reperfusion (Smith et al, 1989). In the investigation of Nguyen (Nguyen et al, 2005) it was concluded that neutrophils play a significant role in injuring cell membranes reloading following periods of unloading, and that this membrane damage was mediated by MPO. A substantial, synergistic effect on the level of muscle lysis when both mechanical loading and neutrophils were applied to muscle cells in vitro was found.…”