Mitochondrial Damage Associated Molecular Patterns From Femoral Reamings Activate Neutrophils Through Formyl Peptide Receptors and P44/42 MAP Kinase

Abstract

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" (DAMPs) like formylated peptides that can activate immunocytes. We therefore studied whether fracture reaming releases mitochondrial DAMPs (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 (mtDNA). Mitochondria were isolated from the residual reaming slurry, sonicated and spun at 12,000g. The resultant MTD were assayed for their ability to cause neutrophil (PMN) Ca 2+ transient production, p44/42 MAPK phosphorylation, IL-8 release and matrix metalloproteinase-9 (MMP9) release with and without formyl peptide receptor-1 (FPR1) blockade. Rats were injected with MTD and whole lung assayed for p44/42 activation.Results-mtDNA 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 2+ flux, activated PMN p44/42 MAPK and caused PMN release of IL-8 and MMP9. Responses to MTD were inhibited by FPR1 blockade using Cyclosporin H and anti-FPR1. 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 facture fixation and lung injury. KeywordsInnate immunity; formyl peptides; fractures; neutrophils Acute lung injury and adult respiratory distress syndrome (ALI/ARDS) occur after fractures in a sporadic entity often termed "fat embolism syndrome" (FES). FES is hard to distinguish from ALI/ARDS occurring after sepsis, and may be associated with reamed nailing more than other methods of fixation. Current concepts emphasize that fracture hematomas are rich in inflammatory mediators 1 -4 that can activate immune cells like neutrophils (PMN) that can injure the lung but it is unknown what the primary events are causing fractures to be rich in mediators. Understanding the events linking mechanical injury to immune organ dysfunction is essential if effective therapies are to be developed. MATERIALS AND METHODS Research complianceStudies were performed under the supervision of the Institutional Review Board (IRB) of Beth Israel Deaconess Medical Center (BIDMC) and Harvard Medical School. Fracture reaming specimens were collected under waiver of consent for discarded materials. Consent was obtained for sampling and archiving of trauma plasma samples from the patients or their legally authorized representative whenever such consent was available. Animal experimentation was approved by the IACUC of BIDMC. Patients and biologic samplesFemoral reamings were collected intra-operati...

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