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Cell‐Free Hemoglobin Increases Lung Alveolar‐Capillary Barrier Permeability Via TLR4
Author(s) -
Shaver Ciara M,
Putz Nathan D,
Bastarache Julie A,
Ware Lorraine B
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.870.5
Subject(s) - bronchoalveolar lavage , lung , tlr4 , vascular permeability , chemistry , inflammation , immunology , cytokine , evans blue , pathology , medicine
Objective Elevated levels of cell‐free hemoglobin (CFH) are present in the airspace of patients with ARDS and are associated with disruption of the alveolar‐capillary barrier. Intra‐tracheal delivery of CFH is sufficient to disrupt alveolar permeability in the lung. However, the mechanisms by which this occurs are unknown. TLR4 expressed on macrophages is a key mediator of the inflammatory effects of CFH, but the role TLR4 in CFH‐induced lung epithelial injury has not been studied. We hypothesized that TLR4 would be a critical mediator of CFH‐induced alveolar‐capillary barrier permeability. Methods TLR4KO or wild‐type (WT) control mice were treated with intra‐tracheal ultrapure endotoxin‐free CFH (100μg). After 24 hours, bronchoalveolar lavage (BAL) was performed. Inflammation was assessed by manual cell counts with differentials and by cytokine expression (ELISA). Alveolar‐capillary barrier function was assessed by BAL protein (BCA assay). Lung epithelial injury was assessed by BAL RAGE (ELISA). For in vitro studies, MLE‐12 mouse lung epithelial cells were treated with CFH (1mg/mL) for 24 hours and paracellular permeability measured by electrical cell‐substrate impedance sensing (ECIS) in the presence or absence of TLR4 inhibitors. Results As compared to WT mice, TLR4KO mice had attenuated alveolar‐capillary barrier disruption in response to CFH as measured by BAL protein (476±71μg/mL vs. 291±58, p<0.001) ( Figure 1). TLR4KO mice also had a 76% reduction in BAL RAGE levels (p<0.001), suggesting that TLR4 mediates lung epithelial cell injury in response to CFH. In addition, TLR4KO had markedly reduced airspace inflammation (p<0.001). To further study whether CFH had direct effects on lung epithelial cells, MLE‐12 cells were exposed to CFH in the presence or absence of TLR4 inhibitors (antibody or small molecules). Whereas CFH caused decreased resistance across the epithelial monolayer, inhibition of TLR4 binding or signaling had no effect on CFH‐mediated permeability in vitro. Conclusions Cell‐free hemoglobin disrupts the alveolar‐capillary barrier in the mouse lung and disrupts paracellular permeability in cultured lung epithelial cell monolayers. TLR4 is a critical mediator of lung epithelial cell injury in vivo and contributes to alveolar‐capillary barrier integrity. Inhibition of TLR4 on epithelial cells did not impact paracellular permeability in vitro. Together, these results demonstrate that CFH has both TLR4‐dependent and TLR4‐independent effects on lung epithelial cells. Support or Funding Information NIH HL103836, HL126671 and HL087738; Vanderbilt Faculty Search Scholars; American Thoracic Society