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" 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.

Figure 1

Quantitative PCR (qPCR) was performed for Cytochrome B in patients with femur fractures or healthy volunteer controls. Cytochrome B is a specific biomarker for the mitochondrial genome. In this analysis, lower PCR cycle values represent more rapid appearance of a detectable signal and thus higher concentration of DNA template in the specimen. Every cycle doubles the amount of DNA. So a difference of 10 cycles represents a 2 fold (i.e. 1024 fold) increase in starting material and so forth.

Figure 2

Human neutrophils (PMN) were prepared for calcium studies by fura-loading in a cuvette with media low in calcium ([Ca²⁺]_o ~50nM). We then added either 10% or 20% FFx supernatant. An immediate intracellular ‘spike’ in [Ca²⁺]_i is seen. Further ‘store-operated’ Ca²⁺ entry is seen on re-calcification of the medium at t=150s. Responses are dose-related. Representative traces are shown, n=3/condition.

Figure 3

Neutrophils were exposed to MTD derived from FFx (MTD) and assayed by western blot for the phosphorylation of p44/42 Mitogen Associated Protein Kinase. DAMPs from FFx rapidly activated this key PMN kinase. Activation was strongly inhibited by cyclosporin H, an inhibitor of the FPR1 receptor. Total p44/42 and β-actin are shown as controls. *P<0.05 (ANOVA/Tukey’s test)

Figure 4

PMN release MMP9 after exposure to FFx MTD. PMN were exposed to FFx MTD (10 min) at the concentrations noted. Supernatants were assayed for MMP-9 by western blot. MTD caused brisk degranulation of MMP-9 (*p< 0.05; ANOVA/Tukey’s test). Release was inhibited by CsH, an inhibitor of FPR1, or by monoclonal antibodies to FPR1 (αFPR1)

Figure 5

PMN produce IL-8 after exposure to MTD from FFx reamings. 10% and 20% MTD caused brisk release of IL-8 at 4 hours (ANOVA p<0.01; post hoc Holm/Sidak p<0.05).

Figure 6

MTD from FFx reamings were injected into rats intravenously. One hour later rats were sacrificed and whole lung homogenates were assayed for activation of p44/42 by western blot. We found brisk phosphorylation of p44/42, suggesting the onset of pulmonary inflammation. *P=0.002 vs naïve (ANOVA/Holm-Sidak).