Patients with the acute respiratory distress syndrome (ARDS) have elevated levels of cell-free hemoglobin (CFH) in the air space, but the contribution of CFH to the pathogenesis of acute lung injury is unknown. In the present study, we demonstrate that levels of CFH in the air space correlate with measures of alveolar-capillary barrier dysfunction in humans with ARDS (r = 0.89, P < 0.001) and in mice with ventilator-induced acute lung injury (r = 0.89, P < 0.001). To investigate the specific contribution of CFH to ARDS, we studied the impact of purified CFH in the mouse lung and on cultured mouse lung epithelial (MLE-12) cells. Intratracheal delivery of CFH in mice causes acute lung injury with air space inflammation and alveolar-capillary barrier disruption. Similarly, in MLE-12 cells, CFH increases proinflammatory cytokine expression and increases paracellular permeability as measured by electrical cell-substrate impedance sensing. Next, to determine whether these effects are mediated by the iron-containing heme moiety of CFH, we treated mice with intratracheal hemin, the chloride salt of heme, and found that hemin was sufficient to increase alveolar permeability but failed to induce proinflammatory cytokine expression or epithelial cell injury. Together, these data identify CFH in the air space as a previously unrecognized driver of lung epithelial injury in human and experimental ARDS and suggest that CFH and hemin may contribute to ARDS through different mechanisms. Interventions targeting CFH and heme in the air space could provide a new therapeutic approach for ARDS.
Background
Systemic blockade of Tissue Factor (TF) attenuates acute lung injury (ALI) in animal models of sepsis but the effects of global TF deficiency are unknown.
Hypothesis
We used mice with complete knockout of mouse TF and low levels (~1%) of human TF (LTF mice) to test the hypothesis that global TF deficiency attenuates lung inflammation in direct lung injury.
Methods
LTF mice were treated with 10 μg of lipopolysaccharide (LPS) or vehicle administered by direct intratracheal (IT) injection and studied at 24 hours.
Results
Contrary to our hypothesis, LTF mice had increased lung inflammation and injury as measured by bronchoalveolar lavage cell count (3.4 × 105 WT LPS versus 3.3 × 105 LTF LPS, p=0.947) and protein (493 μg/ml WT LPS versus 1014 μg/ml LTF LPS, p=0.006), proinflammatory cytokines (TNF-α, IL-10, IL-12, p<0.035 WT LPS versus LTF LPS) and histology compared to wild type mice. LTF mice also had increased hemorrhage and free hemoglobin in the airspace accompanied by increased oxidant stress as measured by lipid peroxidation products (F2-Isoprostanes and Isofurans).
Conclusions
These findings indicate that global TF deficiency does not confer protection in a direct lung injury model. Rather, TF deficiency causes increased intra-alveolar hemorrhage following LPS leading to increased lipid peroxidation. Strategies to globally inhibit tissue factor may be deleterious in patients with ALI.
Tissue factor (TF) initiates the extrinsic coagulation cascade in response to tissue injury, leading to local fibrin deposition. Low levels of TF in mice are associated with increased severity of acute lung injury (ALI) after intratracheal LPS administration. However, the cellular sources of the TF required for protection from LPS-induced ALI remain unknown. In the current study, transgenic mice with cell-specific deletions of TF in the lung epithelium or myeloid cells were treated with intratracheal LPS to determine the cellular sources of TF important in direct ALI. Cell-specific deletion of TF in the lung epithelium reduced total lung TF expression to 39% of wild-type (WT) levels at baseline and to 29% of WT levels after intratracheal LPS. In contrast, there was no reduction of TF with myeloid cell TF deletion. Mice lacking myeloid cell TF did not differ from WT mice in coagulation, inflammation, permeability, or hemorrhage. However, mice lacking lung epithelial TF had increased tissue injury, impaired activation of coagulation in the airspace, disrupted alveolar permeability, and increased alveolar hemorrhage after intratracheal LPS. Deletion of epithelial TF did not affect alveolar permeability in an indirect model of ALI caused by systemic LPS infusion. These studies demonstrate that the lung epithelium is the primary source of TF in the lung, contributing 60-70% of total lung TF, and that lung epithelial, but not myeloid, TF may be protective in direct ALI.Keywords: coagulation; fibrin; pulmonary; acute respiratory distress syndrome; alveolar capillary barrier permeability
Clinical RelevanceThis study evaluates the role of tissue factor (TF) in direct acute lung injury using cell-specific genetic approaches. We identify the lung epithelium as the major source of TF in the airspace. Loss of epithelial TF increases lung injury, impairs coagulation, results in lung hemorrhage, and disrupts alveolar-capillary barrier permeability. These findings identify a potential new target for treatment of severe lung injury in humans.A pathologic hallmark of severe acute lung injury (ALI) in humans is intra-alveolar fibrin deposition, forming hyaline membranes lining the airspace (1). Activation of the tissue factor (TF) pathway is a major driver of coagulation in the airspace (2-5). We previously demonstrated a critical role for TF in protection from ALI caused by intratracheal LPS administration (2). LTF mice, which lack murine TF and express 1% of endogenous levels of human TF to overcome embryonic lethality, developed more severe ALI in response to intratracheal LPS than littermate controls. LTF mice had a local coagulation defect in the airspace, increased histologic lung injury, increased alveolar-capillary
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