title: Mitochondrial damage associated molecular patterns from femoral reamings activate neutrophils through formyl peptide receptors and P44/42 MAP kinase.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA. cjhauser@bidmc.harvard.edu
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Hauser
authors.forename: Tolga
authors.initials: T
authors.lastname: Sursal
authors.forename: Edward K
authors.initials: EK
authors.lastname: Rodriguez
authors.forename: Paul T
authors.initials: PT
authors.lastname: Appleton
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhang
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Itagaki
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D016572:Cyclosporine / Q000494:pharmacology; D004272:DNA, Mitochondrial / Q000276:immunology; D005264:Femoral Fractures / Q000601:surgery*; D006801:Humans; D008297:Male; D020784:Matrix Metalloproteinase 8 / Q000378:metabolism; D008875:Middle Aged; D008928:Mitochondria / Q000235:genetics / Q000276:immunology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D044042:Receptors, Formyl Peptide / Q000276:immunology* / Q000378:metabolism; D055815:Young Adult
chemical_list: D004272:DNA, Mitochondrial; C469986:FPR1 protein, human; D044042:Receptors, Formyl Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016572:Cyclosporine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020784:Matrix Metalloproteinase 8; C050025:cyclosporin H