Trx is a ubiquitous thiol protein, extracellular levels of which are increased under conditions of oxidxative stress and inflammation. In this study we have shown that levels of Trx are raised in BAL fluid and plasma from patients with established ALI/ARDS compared with controls. The increase in BAL fluid levels of Trx correlated with increased levels of IL‐8, a cytokine implicated in the pathogenesis of ALI. When patients with ALI/ARDS were subdivided according to the aetiology of disease, BAL fluid levels of Trx were significantly higher in those with a direct (pulmonary) insult. Moreover, IL‐8 levels and neutrophil counts were also highest in BAL fluid from patients with a pulmonary insult. This is the first study to report extracellular levels of Trx in ALI/ARDS, and to show significant differences depending upon the nature of the lung injury.
Patients recruited for this study had a median duration of lung injury of 12.5 days, reflecting the nature of our institution as a tertiary referral centre to which patients are transferred from other intensive care units. Despite this unavoidable limitation, all our patients still met American/European consensus guidelines for ALI/ARDS at bronchoscopy and all remained sufficiently hypoxaemic to require mechanical ventilation (median duration of ventilation until collection of BAL fluid 13 days for survivors and 11 days for non‐survivors; total median duration of ventilation 50 days for survivors and 34 days for non‐survivors). Moreover, their BAL fluid levels of IL‐8 remained within the range described for patients with ALI/ARDS, suggesting that alveolar inflammation continued. We therefore suggest that our conclusions relating to patients with established ALI/ARDS are valid. Our choice of a control population (self‐ventilating healthy adults) was largely dictated by ethical considerations as objections were raised in relation to the collection of BAL fluid from other critically ill, ventilated patients for use as control samples.
Over the last decade extracellular levels of Trx have been measured in a wide array of conditions characterised by inflammation and oxidative stress, including viral infection, autoimmune disease, heart disease, and ischaemia‐reperfusion injury.11,12
These studies report raised levels in all cases. In keeping with these studies, the results presented here show raised Trx levels in BAL fluid and plasma from patients with ALI/ARDS. However, a previous study showed that serum levels of Trx measured in patients with chronic hepatitis type B were not increased compared with healthy controls.26
This finding therefore reinforces the importance of measuring changes in Trx levels in a given condition as an essential part of the investigation of its role in disease.
Previous reports of serum/plasma levels of Trx suggest that levels in healthy volunteers are in the range 20–30 ng/ml, and become elevated to about 100 ng/ml in various oxidative/inflammatory stress associated disorders.27
These findings are similar to those shown in the current study. The only other published study of BAL fluid levels of Trx reported a median level of 122.6 ng/ml in patients with pulmonary sarcoidosis.28
This compares with 61.6 ng/ml in our ALI/ARDS patients in which a similar method of BAL fluid collection was employed. However, the actual concentration of Trx in the alveolar space is likely to be considerably in excess of that measured in BAL fluid due to the dilutional effects of lavage techniques, usually calculated to be in the order of 50‐fold. If correct, alveolar Trx levels in ALI/ARDS must be between 1 and 7 μg/ml. Even without considering a dilutional effect, levels of Trx were higher in BAL fluid than in plasma in most of the patients in our study. This suggests (allowing for dilution) that BAL fluid levels of Trx in patients with ALI/ARDS are approximately 100‐fold greater than those in plasma. It is therefore unlikely that leakage from plasma to BAL fluid accounts for Trx levels in BAL fluid, unless a specific concentrating mechanism is involved, and suggests that the source of Trx is local to the lung.
Immunostaining with anti‐Trx antibody of biopsy or autopsy tissue from patients with ALI/ARDS and controls showed strong reactivity in alveolar macrophages, type II epithelial cells, bronchial epithelial cells, and interstitial macrophages. We show, for the first time, that staining was significantly greater in alveolar macrophages and type II epithelial cells in ALI/ARDS compared with control tissue. Thus, the stronger immunoreactivity for Trx in ALI/ARDS tissue corresponds with higher levels of Trx in BAL fluid from these patients. A similar finding has been reported in patients with sarcoidosis in which tissue and BAL fluid levels of Trx were higher than in controls.28
In this study, positive staining was seen in macrophages, epithelioid cells, and Langhans' type giant cells. Whether increased staining represents induction of expression or an increase in uptake of Trx is unclear. Assessment of cellular mRNA levels would be necessary to make this distinction and we had insufficient specimens to make this measurement.
The correlation between BAL fluid levels of Trx and IL‐8 suggests a link between extracellular Trx and the inflammatory response that characterises this condition. Whether Trx and IL‐8 are causally linked or whether the relationship simply reflects the intensity of the inflammatory response is unclear. The expression and release of IL‐8 from a variety of primary cells and cell lines is known to be upregulated by exposure to extracellular Trx used at concentrations between 2 and 20 μg/ml.29,30
Taking into account the dilutional effects, these are achievable levels in the alveolar space.
Despite the correlation between BAL fluid concentrations of Trx and IL‐8, and that between IL‐8 and outcome described by others,2
we identified no relationship between Trx levels in plasma and BAL fluid and disease severity or survival. However, results concerning severity should be regarded with caution as only five deaths occurred in the study group. A study comparing serum Trx levels with disease severity in asthma also showed no significant differences among subgroups of mild and moderate asthma sufferers.31
By contrast, a study in patients with chronic heart failure showed that Trx levels increased in proportion to the severity of disease (NYHA class III > NYHA class II) and the degree of impairment of left ventricular function.32
ALI/ARDS precipitated by pulmonary and non‐pulmonary insults are thought increasingly to represent distinct pathophysiological processes.8,9,10
Trx levels in BAL fluid were significantly higher in patients with pulmonary ALI/ARDS than in those with a non‐pulmonary cause. Similarly, IL‐8 levels and neutrophil counts were higher in patients with a direct pulmonary insult. As the median time between insult and collection of BAL fluid was the same for pulmonary and non‐pulmonary groups, this is unlikely to account for the differences between the groups. Moreover, APACHE II, SOFA, and LIS scores were not significantly different between the groups, suggesting that a difference in Trx levels is unlikely to be explained simply in terms of differences in severity of injury. Instead, these differences could represent a more intense inflammatory response in the alveolar space following direct pulmonary insults. The recognition of two forms of ARDS—pulmonary and non‐pulmonary—may lead to improved clinical management. Recognition of differences in disease markers may help in making this distinction.
In summary, Trx levels were raised in BAL fluid and plasma in patients with ALI/ARDS with established lung injury and BAL fluid levels were higher in those in whom the insult was direct. Levels of IL‐8 and neutrophil counts were also higher. Further studies are warranted to determine whether other biochemical/cellular disease markers are significantly greater in patients with direct versus indirect lung injury, and to further elucidate the role of Trx in ALI/ARDS.