Editorial FocusThinking outside the cell: how cell-free hemoglobin can potentiate acute lung injuryJulie A. Bastarache, L. Jackson Roberts II, and Lorraine B. WareJulie A. BastaracheDivision of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; , L. Jackson Roberts IIDepartments of Pharmacology and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; and , and Lorraine B. WareDivision of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TennesseePublished Online:01 Feb 2014https://doi.org/10.1152/ajplung.00355.2013This is the final version - click for previous versionMoreSectionsPDF (38 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations intra-alveolar hemorrhage is a pathological feature of acute lung injury (ALI) that was recognized in the first description of the acute respiratory distress syndrome (ARDS) by Ashbaugh and colleagues in 1967 (2). Over the past four decades, the presence of red blood cells in the air space has largely been considered to be a marker of vascular and tissue injury that is of little mechanistic significance. However, new data suggest that intra-alveolar hemorrhage may contribute to the pathophysiology of lung injury in ARDS and other lung diseases through the release of cell-free hemoglobin into the air spaces. Levels of cell-free hemoglobin are elevated in the air space in patients with ARDS compared with critically ill patients with hydrostatic pulmonary edema (4) and instillation of red blood cells or cell-free hemoglobin causes lung injury in rats (8). Furthermore, in a model of ALI in mice with tissue factor deficiency (4), intra-alveolar hemorrhage was associated with high levels of intra-alveolar cell-free hemoglobin, more severe ALI, and increased lipid peroxidation in the lung. Despite these observations, the cellular and molecular mechanisms by which cell-free hemoglobin in the air space may mediate or potentiate ALI are not well understood. In a recent issue of the Journal, Mumby et al. (13) describe one mechanism by which cell-free hemoglobin may be injurious to the lung epithelium.It is well accepted that intravascular cell-free hemoglobin is important in the pathogenesis of several diseases, including pulmonary hypertension associated with sickle cell disease (17), adverse reactions to red blood cell transfusions (3), and cerebral malaria (9), and as a disease modifier in atherosclerosis (14). Emerging evidence also suggests a potential role for cell-free hemoglobin in the pathophysiology of severe sepsis (1, 10, 12). The common feature of all of these disorders is endothelial dysfunction caused by cell-free hemoglobin. The vascular effects of cell-free hemoglobin are mediated through two main mechanisms: depletion of nitric oxide by direct binding to free hemoglobin and generation of free radicals through hemoglobin peroxidation reactions (16). Both of these molecular mechanisms depend on the iron moiety of cell-free hemoglobin or its associated heme proteins.In the current study, Mumby and colleagues demonstrate that the effects of free hemoglobin on the lung epithelium are not modulated by the iron component of hemoglobin. They report that methemoglobin but not oxyhemoglobin treatment of lung epithelial type II-like cells (A549) stimulates release of IL-8 and MCP-1 through an NF-κB-dependent mechanism. They also show that IL-8 release requires activation of ERK and MCP-1 release requires activation of JNK. Interestingly, antioxidants and iron chelators were not effective in blocking the effects of methemoglobin, suggesting that reactive oxygen species (ROS) and iron are not required for methemoglobin effects. These findings are in distinct contrast to studies of the effects of free hemoglobin on other cell types, which implicate either ROS generation (5, 11) or nitric oxide scavenging (6, 17) as the mechanism of cellular effects. However, the possibility of insufficient duration of exposure to antioxidants and iron chelators prior to treatment with hemoglobin should be noted. Suppression of ROS in humans requires high doses of antioxidants (vitamin E) given for long periods of time (16 days) before an effect is seen (15). The 1-h pretreatment in these experiments may have been insufficient to suppress hemoglobin effects.The report by Mumby and colleagues implies that the effects of cell-free hemoglobin on the lung epithelium are mediated through direct molecular interactions such as cell surface receptor binding rather than nontargeted redox reactions that occur in the vascular space to potentiate ROS-mediated damage. Hemoglobin is known to bind to cell surface receptors in a variety of contexts. CD163 is the hemoglobin scavenging receptor on macrophages, and binding of hemoglobin-haptoglobin complexes to the CD163 receptor initiates degradation of hemoglobin as part of normal homeostatic hemoglobin cycling. However, the authors did not find evidence of CD163 on lung epithelial cells. Hemoglobin can also bind to TLR4 (7) on macrophages and induce cytokine secretion. In the current study, activation of the NF-κB pathway in lung epithelial cells in response to hemoglobin suggests that this could be the mechanism of action in the current study, but the TLR4 pathway was not specifically investigated. Exactly how hemoglobin exerts proinflammatory effects on lung epithelial cells leading to proinflammatory chemokine and cytokine release remains to be determined.Although the iron and heme components were not responsible for the effects of hemoglobin in lung epithelial cells, these effects were specific to methemoglobin rather than oxyhemoglobin; the only difference between methemoglobin and oxyhemoglobin is the redox state of the iron. This finding is puzzling but suggests that perhaps the conformational change of the hemoglobin molecule as the iron is converted from the ferrous 2+ state to the ferric 3+ through oxidation by hydrogen peroxide or oxygen radicals affects its ability to interact with cellular receptors. Another possible explanation for this finding is that further oxidation of methemoglobin to the ferryl species drives oxidative injury in this model that is not inhibited by the antioxidants that were studied. Although the specific hemoglobin species in the air space has never been measured, the highly prooxidant environment in the lung in ALI is likely to support oxidation of the iron moiety of hemoglobin and favor the generation of methemoglobin.The article by Mumby and colleagues provides important new insight into one effect of cell-free hemoglobin on the lung epithelium: stimulation of production of proinflammatory chemokines and cytokines. Whether cell-free hemoglobin leads to other detrimental effects on the lung epithelium such as alterations in epithelial permeability or alveolar epithelial fluid transport function is an important area of future study. Beyond the effects of cell-free hemoglobin on the lung epithelium, there are several important but as yet unanswered questions about the role of intra-alveolar cell-free hemoglobin in lung disease that are raised by this article. First, the mechanisms that regulate liberation and persistence of free hemoglobin in the air space are unknown. It will be important to determine whether free hemoglobin is released from red blood cells in the air space or leaks in from the plasma as a result of increased permeability, particularly in light of reports of increased circulating levels of free hemoglobin in patients with sepsis (10). In addition, studies to determine the mechanisms of clearance of free hemoglobin from the air space and the impact of ALI and other lung diseases on these pathways are needed. Another important question is What is the redox state of cell-free hemoglobin in the air space? Is hemoglobin oxidized to methemoglobin or to the ferryl form in the air space? Finally, are these findings clinically relevant to ARDS and other lung diseases and, if so, does intra-alveolar free hemoglobin represent a novel therapeutic target? Additional studies in patients with ARDS and other lung disorders will be needed to answer these questions. The current study is an important first step toward advancing our understanding of the role of cell-free hemoglobin in human lung disease. The impact of these studies is likely to be far reaching, opening up a new field of inquiry into a novel pathogenic mechanism of epithelial injury in lung disease.GRANTSThis work was supported by NIH HL103836, HL112656, and HL090785, and an American Heart Association Established Investigator AwardDISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the author(s).AUTHOR CONTRIBUTIONSJ.A.B. and L.B.W. drafted manuscript; J.A.B., L.J.R., and L.B.W. edited and revised manuscript; J.A.B., L.J.R., and L.B.W. approved final version of manuscript.REFERENCES1. Adamzik M, Hamburger T, Petrat F, Peters J, de Groot H, Hartmann M. Free hemoglobin concentration in severe sepsis: methods of measurement and prediction of outcome. 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Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Cited ByA comparison of different methods of red blood cell leukoreduction and additive solutions on the accumulation of neutrophil‐priming activity during storage1 September 2018 | Transfusion, Vol. 58, No. 8There is blood in the water: hemolysis, hemoglobin, and heme in acute lung injuryAmit Gaggar and Rakesh P. Patel1 October 2016 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 311, No. 4 More from this issue > Volume 306Issue 3February 2014Pages L231-L232 Copyright & PermissionsCopyright © 2014 the American Physiological Societyhttps://doi.org/10.1152/ajplung.00355.2013PubMed24337924History Received 6 December 2013 Accepted 9 December 2013 Published online 1 February 2014 Published in print 1 February 2014 Metrics