Mitochondrial damage associated molecular patterns from femoral reamings activate neutrophils through formyl peptide receptors and P44/42 MAP kinase.

HYPOTHESIS Fractures and femoral reaming are associated with lung injury. The mechanisms linking fractures and inflammation are unclear, but tissue disruption might release mitochondria. Mitochondria are evolutionarily derived from bacteria and contain "damage associated molecular patterns" like formylated peptides that can activate immunocytes. We therefore studied whether fracture reaming releases mitochondrial damage associated molecular patterns (MTD) and how MTD act on immune cells. METHODS Femur fracture reamings (FFx) from 10 patients were spun to remove bone particulates. Supernatants were assayed for mitochondrial DNA. Mitochondria were isolated from the residual reaming slurry, sonicated, and spun at 12,000 g. The resultant MTD were assayed for their ability to cause neutrophil (PMN) Ca transient production, p44/42 MAPK phosphorylation, interleukin-8 release, and matrix metalloproteinase-9 release with and without formyl peptide receptor-1 blockade. Rats were injected with MTD and whole lung assayed for p44/42 activation. RESULTS Mitochondrial DNA appears at many thousand-fold normal plasma levels in FFx and at intermediate levels in patients' plasma, suggesting release from fracture to plasma. FFx MTD caused brisk PMN Ca flux, activated PMN p44/42 MAPK, and caused PMN release of interleukin-8 and matrix metalloproteinase-9. Responses to MTD were inhibited by formyl peptide receptor-1 blockade using cyclosporine H or anti-formyl peptide receptor-1. MTD injection caused P44/42 phosphorylation in rat lung. CONCLUSIONS FFx reaming releases mitochondria into the wound and circulation. MTD then activates PMN. Release of damage signals like MTD from FFx may underlie activation of the cytokine cascades known to be associated with fracture fixation and lung injury.