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

Abstract

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.

Keywords: acute respiratory distress syndrome; air space inflammation; hemin; hemoglobin; lung epithelium; permeability.

Fig. 1.

Release of cell-free hemoglobin into the air space is a feature of human and experimental acute lung injury. A: cell-free hemoglobin (CFH) levels are significantly higher in pulmonary edema fluid (EF) compared with plasma (PL) of patients with acute respiratory distress syndrome (ARDS), n = 18; *P = 0.011 vs. control. The levels of CFH in EF from these patients was previously published and are reported here for direct comparison to PL CFH levels (6). B: in patients with ARDS, EF CFH correlates strongly with EF-to-PL protein ratio, an index of alveolar-capillary barrier permeability, rho = 0.89, P < 0.001 by Spearman's correlation coefficient. C: CFH is elevated in bronchoalveolar lavage (BAL) fluid of mice treated with mechanical ventilation ± intratracheal LPS; *P < 0.029 for each group vs. control, n = 3–5 per group. D: BAL CFH correlates strongly with BAL protein in mice with ventilator induced lung injury, rho = 0.89, P < 0.001 by Spearman's correlation. E: BAL CFH is increased during experimental bacterial pneumonia compared with control animals, *P = 0.05, n = 5–9 per group.

Fig. 2.

Cell-free hemoglobin induces acute air space inflammation in mice. Mice were treated with 100 μg endotoxin-free CFH or 100 μg albumin control and sampled after 24 h. CFH increases total BAL fluid cell counts (*P = 0.032 vs. albumin control) (A) and trends toward increased neutrophils in the air space (*P = 0.09 vs. albumin control) (B) compared with control mice. There is no significant difference in numbers of macrophages or lymphocytes between treatments. C: CFH increases proinflammatory cytokine concentrations in BAL fluid; n = 6–8 per group.

Fig. 3.

Cell-free hemoglobin causes histological lung injury and disrupts the alveolar-capillary barrier. A–C: CFH increases the total lung injury score and edema score as assessed by histological examination (*P = 0.033 for edema score and P = 0.002 for total lung injury score compared with PBS control). D: CFH increases BAL protein, consistent with disruption of the alveolar-capillary barrier (*P = 0.013). E: CFH induced injury to epithelial cells as measured by concentration of receptor of advanced glycation end products (RAGE) in BAL fluid (*P = 0.001 vs. control); n = 6–8 in each group.

Fig. 4.

Cell-free hemoglobin induces cytokine production and permeability defects in cultured mouse lung epithelial cells. MLE-12 lung epithelial cells produce higher levels of IL-6 (A) and CXCL-1/KC (B) in response to increasing doses of CFH in vitro (*P < 0.05 vs. control, #P < 0.05 vs. 0.01 mg/ml dose). MLE-12 cells increase IL-6 (C) and KC (D) transcription in response to CFH exposure (*P < 0.05 vs. control, #P < 0.05 vs. 0.01 mg/ml). E: CFH reduces epithelial permeability as measured by electric cell-substrate impedance sensing (ECIS) over 24 h (P < 0.001 vs. control); n = 4–8 in each group.

Fig. 5.

The heme moiety of cell-free hemoglobin affects permeability but not inflammation during acute lung injury. C57Bl/6 mice were treated with intratracheal CFH (100 μg) or hemin (60 μM), the chloride salt of heme, for 24 h prior to sample collection. Hemin causes less alveolar inflammation as measured by bronchoalveolar cell counts (*P = 0.037) (A) with fewer neutrophils in the air space (*P = 0.009) (B). C: hemin induces less proinflammatory cytokines in BAL compared with CFH. BAL protein (D) was equivalent between CFH and hemin (P = 0.178). Hemin caused less release of BAL RAGE (E) compared with CFH (*P = 0.006); n = 8 in each group.

Fig. 6.

The hemin moiety of cell-free hemoglobin affects permeability but not inflammation in cultured lung epithelial cells. MLE-12 cells were incubated with media control, CFH (1 mg/ml), or hemin (60 μM) for 24 h. Expression of IL-6 (A) and KC (B) protein were measured by ELISA. C: permeability was measured by ECIS over time (*P < 0.001 vs. control at 24 h); n = 4–8 replicates for each measurement.