Brandon S. Grove

Low levels of tissue factor lead to alveolar haemorrhage, potentiating murine acute lung injury and oxidative stress

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. 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 injection and studied at 24 h. Results Contrary to our hypothesis, LTF mice had increased lung inflammation and injury as measured by bronchoalveolar lavage cell count (3.4×105 wild-type (WT) LPS vs 3.3×105 LTF LPS, p=0.947) and protein (493 μg/ml WT LPS vs 1014 μg/ml LTF LPS, p=0.006), proinflammatory cytokines (TNF-α, IL-10, IL-12, p<0.035 WT LPS vs LTF LPS) and histology compared with WT mice. LTF mice also had increased haemorrhage and free haemoglobin 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 haemorrhage following LPS leading to increased lipid peroxidation. Strategies to globally inhibit TF may be deleterious in patients with ALI.

Regulation of Alveolar Procoagulant Activity and Permeability in Direct Acute Lung Injury by Lung Epithelial Tissue Factor

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.

Interferon-γ and tumor necrosis factor-α act synergistically to up-regulate tissue factor in alveolar epithelial cells

ABSTRACT Fibrin deposition mediated through activation of tissue factor (TF) in the airspace is central to the pathogenesis of acute lung injury. Defining the mechanisms of TF regulation in the lung is critical to understanding pulmonary fibrin formation. Tumor necrosis factor-α (TNF-α) up-regulates TF in the injured lung, and there is emerging evidence that another cytokine, interferon-γ (IFN-γ), also modulates expression. The effects of TNF-α and IFN-γ on regulation of TF were studied in alveolar epithelial A549 cells. In addition, potential mechanisms of modulation of TF expression by the 2 cytokines were analyzed with the hypothesis that IFN-γ acts synergistically with TNF-α to up-regulate alveolar epithelial TF through modulation of TNF receptor (TNFR) expression. TNF-α but not IFN-γ treatment increased TF mRNA, protein, and cell surface TF activity. The combination of IFN-γ and TNF-α treatment augmented the effects of TNF-α on TF up-regulation and also increased release of procoagulant microparticles (MPs) from A549 cells. IFN-γ modulated expression of both TNF-α receptors. Studies utilizing neutralizing antibodies against the two TNF receptors showed that the TF effects were mediated primarily through augmentation of TNFR1-dependent cellular responses. These findings have important implications for regulation of fibrin formation in the lung in the setting of acute inflammation.

Cell-free hemoglobin: a novel mediator of acute lung injury

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.

Myeloid tissue factor does not modulate lung inflammation or permeability during experimental acute lung injury.

Tissue factor (TF) is a critical mediator of direct acute lung injury (ALI) with global TF deficiency resulting in increased airspace inflammation, alveolar-capillary permeability, and alveolar hemorrhage after intra-tracheal lipopolysaccharide (LPS). In the lung, TF is expressed diffusely on the lung epithelium and intensely on cells of the myeloid lineage. We recently reported that TF on the lung epithelium, but not on myeloid cells, was the major source of TF during intra-tracheal LPS-induced ALI. Because of a growing body of literature demonstrating important pathophysiologic differences between ALI caused by different etiologies, we hypothesized that TF on myeloid cells may have distinct contributions to airspace inflammation and permeability between direct and indirect causes of ALI. To test this, we compared mice lacking TF on myeloid cells (TF∆mye, LysM.Cre+/−TFflox/flox) to littermate controls during direct (bacterial pneumonia, ventilator-induced ALI, bleomycin-induced ALI) and indirect ALI (systemic LPS, cecal ligation and puncture). ALI was quantified by weight loss, bronchoalveolar lavage (BAL) inflammatory cell number, cytokine concentration, protein concentration, and BAL procoagulant activity. There was no significant contribution of TF on myeloid cells in multiple models of experimental ALI, leading to the conclusion that TF in myeloid cells is not a major contributor to experimental ALI.

Novel Method for Noninvasive Sampling of the Distal Airspace in Acute Respiratory Distress Syndrome

Rationale: A major barrier to a more complete understanding of acute respiratory distress syndrome (ARDS) pathophysiology is the inability to sample the distal airspace of patients with ARDS. The heat moisture exchanger (HME) filter is an inline bacteriostatic sponge that collects exhaled moisture from the lungs of mechanically ventilated patients. Objectives: To test the hypothesis that HME filter fluid (HMEF) represents the distal airspace fluid in patients with ARDS. Methods: Samples of HMEF were collected from 37 patients with acute pulmonary edema (either from ARDS or hydrostatic causes [HYDRO; control subjects]). Concurrent undiluted pulmonary edema fluid (EF) and HMEF were collected from six patients. HMEF from 11 patients (8 ARDS and 3 HYDRO) were analyzed by liquid chromatography‐coupled tandem mass spectometry. Total protein (bicinchoninic acid assay), MMP‐9 (matrix metalloproteinase‐9), and MPO (myeloperoxidase) (ELISA) were measured in 29 subjects with ARDS and 5 subjects with HYDRO. SP‐D (surfactant protein‐D), RAGE (receptor for advanced glycation end‐products) (ELISA), and cytokines (IL‐1&bgr;, IL‐6, IL‐8, and tumor necrosis factor‐&agr;) (electrochemiluminescent assays) were measured in six concurrent HMEF and EF samples. Measurements and Main Results: Liquid chromatography‐coupled tandem mass spectrometry on concurrent EF and HMEF samples from four patients revealed similar base peak intensities and m/z values indicating similar protein composition. There were 21 significantly elevated proteins in HMEF from patients with ARDS versus HYDRO. Eight proteins measured in concurrent EF and HMEF from six patients were highly correlated. In HMEF, total protein and MMP‐9 were significantly higher in ARDS than in HYDRO. Conclusions: These data suggest that HMEF is a novel, noninvasive method to accurately sample the distal airspace in patients with ARDS.