These studies demonstrate that EphA2 contributes to the pathophysiology of experimental acute lung injury. The expression in lung tissue of both the EphA2 receptor and its principal ligand, ephrin-A1, is increased during the early stages of bleomycin-induced lung injury. More importantly, EphA2-deficient animals are protected from such injury. Mechanistically, acute lung injury is associated with changes in both vascular permeability and the recruitment of inflammatory cells and generation of chemokines. EphA2-deficient animals appear to be protected from both the permeability and the inflammatory changes associated with this experimental injury. These findings extend previous work that described a role for EphA2 in the regulation of pulmonary vascular permeability, and they constitute one of the first demonstrations of a role for EphA2 in the regulation of inflammatory responses to tissue injury.
The role of EphA2 in the postnatal lung remains poorly understood. We previously reported that the stimulation of EphA receptors in the pulmonary vasculature with the exogenous soluble ephrin-A1 ligand leads to increases in albumin extravasation into the lung (4
). Although EphA2 is the most highly expressed EphA receptor in lung endothelial cells, the effect of ephrin-A1 on lung vascular permeability had not been shown definitively to require EphA2. Using the isolated, perfused lung approach in the present study, we confirmed that an infusion of ephrin-A1 ligand into the pulmonary vasculature increases vascular permeability, as measured by the filtration coefficient (Kf). In addition, the permeability response to ephrin-A1 was lost in EphA2-knockout mice, demonstrating that EphA2 transduces the permeability-increasing effect of ephrin-A1 in the pulmonary circulation. These results are consistent with our previously published work, and extend those findings to a second species. This effect of the ligand stimulation of EphA2 on vascular permeability suggests the hypothesis that changes in the expression of EphA2 could contribute to pathologic situations associated with increased vascular permeability, such as acute lung injury and sepsis.
Acute lung injury is a common and severe clinical problem in both adults and children. The acute lung injury caused by an instillation of bleomycin into the airways comprises a model that recapitulates several key features of human acute lung injury, most importantly an early edematous phase characterized by increased permeability and neutrophilic inflammatory cell influx (7
). As a result of these parallels with human lung injury, we studied the role of EphA2 in the early stages of bleomycin injury. Our results showed a clear and substantial increase in the expression of EphA2 in the distal lung of wild-type mice after bleomycin injury, localized to the alveolar septae and alveolar macrophages. These findings are consistent with previous results showing an increased expression of lung EphA2 after other insults, such as injections of LPS or exposure to hypoxia after a viral respiratory infection (5
). Of even greater interest, the response of EphA2-deficient animals to bleomycin injury suggests that EphA2 is not only increased in the injured lung, but that it also plays an important role in the development of such injury. Compared with wild-type mice, EphA2 -deficient mice accumulated less lung water and displayed less protein leak into the airspaces after bleomycin injury. These findings provide additional evidence that EphA2 contributes to the disruption of the alveolar–capillary barrier seen in acute lung injury, and are consistent with both our isolated, perfused lung experiments and our previous work describing the pro-permeability effects of the ligand stimulation of EphA2 in the lung endothelium. Although these data, as well as those in previous studies, clearly show that EphA2 is expressed in lung endothelial cells, it is noteworthy that EphA2 is also expressed in numerous other cell types that contribute to acute lung injury, including inflammatory cells and alveolar epithelial cells (11
). Whether EphA2 also modulates lung epithelial permeability is not known, but such an effect is plausible, given that the activation of EphA2 was shown to disrupt adherens junctions in other epithelia (12
The influx of inflammatory cells to the lung is a key element of acute lung injury, both in human patients and in our bleomycin model. We were intrigued to find that EphA2-deficient animals accumulated far fewer inflammatory cells and, in particular, neutrophils in their lungs in response to injury than did wild-type animals, suggesting that EphA2 contributes not just to changes in permeability but also to inflammatory responses. Chemokines are known as key mediators of leukocyte recruitment to the injured lung, and the altered elaboration of chemokines in EphA2-deficient animals provided a possible explanation for our findings. Indeed, concentrations in lung tissue of important neutrophil (KC/CXCL1) and monocyte (MCP1/CCL2) chemoattractants were markedly lower in EphA2-deficient mice after injury than in control animals. These results suggest a previously unrecognized contribution of ephrin signaling to the generation of chemokines and recruitment of leukocytes in the lung, in addition to effects on endothelial permeability. Whether EphA2 also contributes to the transmigration and retention of recruited leukocytes is not certain, although reduced permeability in EphA2-deficient vessels might also be expected to impede the transmigration of leukocytes. Mechanistically, the production of chemokines is generally regulated by the activation of proinflammatory transcription factors. The finding that the ligand stimulation by ephrin-A1 of endothelial cells triggers the activation of the prototypical proinflammatory transcription factor NF-κβ as well as the mRNA expression of CXCL1, CCL2, and ICAM1 is consistent with our observations on the expression of chemokines in intact animals. These results suggest that ligand stimulation of EphA2 has the capacity to regulate inflammatory responses transcriptionally. Whether other cell types respond similarly, and whether other transcription factors in addition to NF-κβ are activated by ephrin-A1, remain to be determined. Of further interest was a trend (that did not reach statistical significance) toward reduced macrophage numbers in the lavage fluid from uninjured EphA2-knockout animals compared with uninjured control animals. This observation, if borne out in future studies, could suggest that EphA2 also regulates leukocyte trafficking in the normal lung as well as in the setting of lung injury.
The mechanism by which EphA2 is activated in the injured lung remains uncertain. As already discussed, one likely possibility involves ligand stimulation. Ephrin-A1 is the principal ligand for EphA2, and it is known to be expressed by endothelial cells, epithelial cells, and some leukocytes (15
). The expression of ephrin-A1 is also known to be induced by some cytokines, consistent with a possible role in inflammation (2
). In support of this idea, we found increased concentrations of ephrin-A1 ligand protein in the injured lungs. Whereas ephrin-A1 principally exists as a membrane-anchored protein, soluble forms (both monomeric and multimeric) have also been described (18
). Our results suggest that such forms of ephrin-A1 are released into the alveolar space in the setting of lung injury. These results are most consistent with the idea that the increased expression of both ephrin-A1 ligand and the EphA2 receptor in the injured lung leads to the increased ligand-mediated activation of EphA2 in that setting. Alternatively, both the ligand-independent effects of EphA2 overexpression and the transactivation of EphA2 by non–ephrin ligands have also been described (including thrombin, another mediator known to be released in large amounts in the injured lung) (21
). Whether EphA2 acts via these additional mechanisms in the setting of lung injury will require further study. Given the demonstrated role of ephrins in the nervous system as key regulators of cell–cell contacts and cell–cell signaling, ephrin ligands and receptors seem likely to play a similar role in the lung, and to regulate responses to tissue injury via their role in cell-to-cell communication.
In conclusion, we demonstrate that EphA2 regulates permeability and inflammation in the injured lung, and that EphA2 thus contributes to the pathophysiology of acute lung injury. The mechanisms by which EphA2 regulates lung inflammation and leak and the possible role of EphA2 as a therapeutic target in the injured lung deserve further investigation.