In the United States, lungs of approximately 75% eligible donors are ineligible for transplantation . These lungs are an invaluable resource to study lung function and organ preservation to increase eligibility for transplantation. In this study, explanted human lungs rejected for transplantation underwent ex vivo lung perfusion and a detailed characterization of perfusate and airspace biomarker kinetics was performed under 6 experimental conditions.
During 4 hours of EVLP, we found that the lung has a remarkable capacity to produce proteins associated with immune (IL-6, IL-8, sTNFR1) and endothelial (Ang-2) responses. The baseline level and the change in concentration of these mediators after 4 hours of ex vivo perfusion was unrelated to the duration of cold ischemia time and parameters associated with deranged lung epithelial (alveolar fluid clearance) or endothelial (percent weight gain) function . The addition of fresh whole blood attenuated the increase in perfusate IL-6 in lungs exposed to intravenous bacteria. The addition of a lethal dose of Gram-positive bacteria (S. pneumoniae) did not significantly change perfusate or airspace biomarker kinetics, with the exception of perfusate IL-6 in the absence of exogenous blood.
A similar high rise in some biomarkers quantified in this study has been demonstrated in prior reports of EVLP [20, 28]. IL-6 and IL-8 are inflammatory cytokines traditionally synonymous with organ injury and poor outcome. In the EVLP model, several studies of cross-sectional cytokine levels suggest that donor lung levels of these biomarkers are inversely associated with graft function in the transplant recipient [17–20]. Interestingly, cytokines in the EVLP perfusate of successfully transplanted lungs also reach extremely high levels (as much as a 30–100 fold increase in IL-6 and IL-8) without evidence of primary graft dysfunction (PGD) [20, 21]. If IL-6 and IL-8 are always injurious, why do not more transplant recipients develop PGD?
High levels of IL-6, IL-8, sTNFR1, and Ang-2 in plasma samples of patients with the acute respiratory distress syndrome (ARDS) are well known for their association with poor outcomes [29, 30]. However, plasma biomarker levels are substantially lower than those reported in EVLP perfusate. This discrepancy between the extremely high levels of inflammatory cytokines in the EVLP perfusate and the lack of injury in every transplant recipient as well as the lack of association with experimental measurements of injury in our study (percent weight gain, final AFC) suggest that these cytokines have an additional biologic significance , especially in the EVLP model.
The source of biomarker production and the mechanism of accumulation are uncertain. The EVLP model lacks mechanisms associated with clearance, specifically the liver , the kidneys , and components of the vascular compartment, the absence of which may explain the striking difference between biomarker concentrations detectable in plasma relative to the perfusate and airspaces of the ex vivo perfused lung. In our experiments in which only 100 ml of fresh whole blood was added to 2L of EVLP perfusate (representative of a hematocrit of approximately 2%), there appeared to be a trend toward a dampening effect on the increase in perfusate IL-6 and IL-8 levels in control lungs as well as in lungs exposed to IV S. pneumoniae. This suggests that protective factors are present in blood that either reduce the production of these cytokines, that increase their enzymatic clearance, or that facilitate their sequestration . Future investigation of whole blood components that may be responsible for the decrease in inflammatory biomarker levels may be relevant to transplantation as several studies support the notion that high IL-8 in perfusate is related to an increased incidence of PGD3 in the recipient [17–19].
A potential source of cytokines may be cell necrosis and apoptosis due to ischemia-reperfusion injury . However, neither the baseline nor the change in biomarkers after 4 hours of EVLP was associated with cold ischemia time, suggesting that presumed cell necrosis and apoptosis cannot on their own explain the high abundance of cytokines in our model. In fact, prolonged hypothermia without reperfusion did not increase pneumocyte apoptosis in a rat lung transplant model . Also, there is evidence from studying tissue biomarker levels in the human EVLP model that inflammatory cytokine levels do not significantly differ after extended cold ischemia time .
Of note, not all biomarker levels increased to the same extend during EVLP. The small change in Ang-2 levels during the 4 hours of ex vivo perfusion relative to inflammatory biomarker levels is puzzling. This protein is produced by the endothelium and is stored in Weibel Palade bodies , where it has a long half-life (over 18 hours) and can be secreted within minutes of stimulation . However, despite lung injury due to ischemia-reperfusion and addition of lethal doses of S. pneumoniae, the magnitude of Ang-2 change was minimal compared to the change in IL-6 and IL-8.
The observation that starting AFC is only significantly associated with baseline IL-6 and IL-8 levels in the airspace compartment is intriguing. It is plausible that when the lung epithelial function is intact and alveolar fluid is clearance is preserved , IL-6 and IL-8 may concentrate in the airspaces. It also implies that when interpreting levels of these cytokines in the airspace compartment of the EVLP model, it is important to measure and account for lung alveolar fluid clearance.
This study has some limitations. First, only two time points were studied during the course of perfusion, and as such, it is possible we missed important timepoint biomarker production trends, particularly in experimental conditions in the presence of bacteria. It is also possible that the perfusion was not long enough to appreciate differences between the experimental conditions included in this study. Secondly, we studied only four soluble proteins and to detect relevant differences between experimental conditions, other proteins may need to be studied. It is also plausible that other metrics, such as RNA expression, microvesicle or lipid production, could provide insight into biological differences between the experimental conditions. Third, our experiments were performed with a single bacterial pathogen, and as such, the data may not be extrapolated to infection with other pathogens.