Trauma and sepsis can cause acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) in part by triggering neutrophil (PMN)-mediated increases in endothelial cell (EC) permeability. We had shown that mitochondrial (mt) damage-associated molecular patterns (DAMPs) appear in the blood after injury or shock and activate human PMN. So we now hypothesized that mitochondrial DAMPs (MTD) like mitochondrial DNA (mtDNA) and peptides might play a role in increased EC permeability during systemic inflammation and proceeded to evaluate the underlying mechanisms. MtDNA induced changes in EC permeability occurred in two phases: a brief, PMN-independent ‘spike’ in permeability was followed by a prolonged PMN-dependent increase in permeability. Fragmented mitochondria (MTD) caused PMN-independent increase in EC permeability that were abolished with protease treatment. Exposure to mtDNA caused PMN-EC adherence by activating expression of adherence molecule expression in both cell types. Cellular activation was manifested as an increase in PMN calcium flux and EC MAPK phosphorylation. Permeability and PMN adherence were attenuated by endosomal TLR inhibitors. EC lacked formyl peptide receptors but were nonetheless activated by mt-proteins, showing that non-formylated mt-protein DAMPs can activate EC. Mitochondrial DAMPs can be released into the circulation by many processes that cause cell injury and lead to pathologic endothelial permeability. We show here that mitochondria contain multiple DAMP motifs that can act on EC and/or PMN via multiple pathways. This can enhance PMN adherence to EC, activate PMN-EC interactions and subsequently increase systemic endothelial permeability. Mitochondrial DAMPs may be important therapeutic targets in conditions where inflammation pathologically increases endothelial permeability.

Bacterial DNA (bDNA) contains hypo-methylated “CpG” repeats that can be recognized by toll-like receptor (TLR)-9 as a pathogen-associated molecular pattern (PAMP). The ability of bDNA to initiate lung injury via TLR-9 has been inferred on the basis of studies using artificial CpG DNA. But the role of authentic bDNA in lung injury is still unknown. Moreover, the mechanisms by which CpG DNA species can lead to pulmonary injury are unknown, although neutrophils (PMN) are thought to play a key role in the genesis of septic acute lung injury (ALI). We evaluated the effects of bDNA on PMN-endothelial cell (EC) interactions thought critical for initiation of ALI. Using a bio-capacitance system to monitor real-time changes in endothelial permeability, we demonstrate here that bDNA causes EC permeability in a dose-dependent manner uniquely in the presence of PMN. These permeability changes are inhibited by chloroquine, suggesting TLR9-dependency. When PMN were pre-incubated with bDNA and applied to EC or when bDNA was applied to EC without PMN, no permeability changes were detected. To study the underlying mechanisms we evaluated the effects of bDNA on PMN-EC adherence. bDNA significantly increased PMN adherence to EC in association with up-regulated adhesion molecules in both cell types. Taken together, our results strongly support the conclusion that bDNA can initiate lung injury by stimulating PMN-EC adhesive interactions predisposing to endothelial permeability. bDNA stimulation of TLR9 appears to promote enhanced gene expression of adhesion molecules in both cell types. This leads to PMN-EC cross-talk which is required for injury to occur.