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Ann Thorac Surg 2006;81:1844-1850
© 2006 The Society of Thoracic Surgeons
a Department of Surgery, Division of Thoracic and Cardiovascular Surgery, University of Florida, Gainesville, Florida
b Department of Medicine, Division of Pulmonology, University of Florida, Gainesville, Florida
c Department of Radiology, Division of Thoracic Radiology, University of Florida, Gainesville, Florida
d Department of Surgery, Laboratory of Inflammation Biology and Surgical Science, University of Florida, Gainesville, Florida
Accepted for publication November 28, 2005.
* Address correspondence to Dr Beaver, Division of Thoracic and Cardiovascular Surgery, PO Box 100286, Gainesville, FL 32610-0286 (Email: beavetm{at}surgery.ufl.edu).
Presented at the Forty-first Annual Meeting of The Society of Thoracic Surgeons, Tampa, FL, Jan 24–26, 2005.
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Abstract |
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METHODS: Pulmonary arterial and systemic arterial samples were obtained at baseline and at 4, 8, and 24 hours after reperfusion. Post–lung transplant reperfusion-edema and PGF were defined as PaO2/FiO2 less than 300 with a mild or moderate infiltrate, or less than 200 with a severe infiltrate and ventilator dependence after 72 hours, respectively. Tumor necrosis factor alpha (TNF
), interleukin (IL)-6, IL-8, and IL-10 concentrations were determined by immunoassay.
RESULTS: Fifteen single and 6 bilateral lung recipients were studied. Six (29%) had PLTRE and 4 (19%) had PGF; these patients had an overall elevation in plasma IL-6, IL-8, and IL-10 concentrations (all p < 0.05). Subgroup analysis revealed a significantly greater elevation in IL-6, IL-8, and IL-10 levels in PGF patients (all p < 0.01) versus PLTRE. In the PGF group, TNF and IL-10 concentrations were significantly greater in the systemic versus the pulmonary arterial samples (p < 0.05).
CONCLUSIONS: Patients with PLTRE and PGF exhibited graded increases in IL-6, IL-8, and IL-10 concentrations. The PGF patients had higher TNF and IL-10 systemic arterial concentrations overall, consistent with the allograft being a source of this cytokine production.
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Introduction |
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Our institution has previously identified associations between the appearance of proinflammatory cytokines in the systemic circulation after thoracoabdominal aortic aneurysm repair and the development of multiple organ failure [6]. In the setting of lung transplantation, Mal and colleagues [7] have examined plasma cytokine concentrations on postoperative days 1 to 7; however, because PLTRE develops soon after reperfusion, the measurements in this study were not reflective of the early cytokine response. For example, interleukin (IL)-8 elevations in lung allograft biopsies at 2 hours have been shown to correlate with later pulmonary dysfunction [8]. And Pham and associates [9] found elevations of IL-6 at 4 hours correlated with prolonged mechanical ventilation, more severe alveolar damage, and poorer 1-month graft survival. The complete nature of the proinflammatory cytokine cascade that evolves after lung transplantation has yet to be elucidated.
The purpose of this study was to examine the relationship between the cytokine response and the development of PLTRE and PGF. We hypothesized that elevations in the proinflammatory cytokines tumor necrosis factor alpha (TNF), IL-6, and IL-8 would be associated with allograft injury as defined by PLTRE and PGF. We further hypothesized that the allograft would be the primary source of these cytokines in the plasma.
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Patients and Methods |
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The cytokines examined included TNF, a proximal mediator in the cytokine cascade; IL-6, a generalized marker of inflammation; IL-8, a chemokine responsible for neutrophil recruitment and activation; and IL-10, a counterregulatory cytokine involved in suppression of the proinflammatory response. Samples were collected in ethylenediamine tetraacetic acid (EDTA), placed immediately on ice, and centrifuged at 3,000 rpm (4°C) for 10 minutes. Supernatant was withdrawn and frozen at –70°C until samples were analyzed for cytokine concentrations using enzyme-linked immunosorbent assay (ELISA) kits (R & D systems, Minneapolis, Minnesota).
Data Analysis
Clinical variables were recorded using a Microsoft Excel database (Microsoft, Redmond, Washington) and included allograft ischemia times, cardiopulmonary bypass, ratios of arterial oxygen pressure tension (PaO2) levels and fraction of inspired oxygen (FiO2); pulmonary artery and systemic hemodynamics; radiographic findings of reperfusion injury; and the number of hours of required mechanical ventilation after lung transplantation.
Analyses of cytokine levels at each time point and for each clinical injury group were conducted using nonparametric two-way analysis of variance. Post-hoc comparisons were performed using Dunn's test. Pulmonary arterial and systemic arterial samples were compared in a paired fashion using Wilcoxon signed rank analysis, in order to compare overall differences in cytokine levels before and after reperfusion of the allograft. All statistical analyses were conducted with SigmaStat, version 3.1 software package (Systat, Richmond, California).
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Results |
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Mean pulmonary arterial pressures within the first postoperative day were significantly different among the three groups (p < 0.01), and specifically higher in the PGF population than in the other two groups (both p < 0.05). Mean cardiac index and systemic vascular resistance were also recorded, with PGF patients having more hemodynamic compromise.
Consistent with our definitions, median mechanical ventilation times were longer in PGF and PLTRE, and distinct among the three groups (p < 0.01). The chest radiograph scoring system also revealed unique infiltrate patterns among the three groups (p < 0.001). Patients with PLTRE and PGF had progressively lower PaO2/FiO2 ratios than those patients without injury (p < 0.05).
Cytokine Results
The cytokine concentrations from pulmonary arterial and systemic arterial samples for the lung transplant recipients from the no injury, PLTRE, and PGF subgroups are displayed in Figures 2 through 4
for IL-6, IL-8, and IL-10, respectively (no noticeable elevation in TNF was identified at the time points measured). Subgroup analysis revealed distinct IL-6, IL-8, and IL-10 patterns in both arterial and venous samples when comparing PGF versus PLTRE (IL-6, both p < 0.01; remainder all p < 0.001), as well as PGF versus patients with no injury (all p < 0.001). Interleukin-6, IL-8, and IL-10 concentrations in patients with PLTRE were higher than in those without injury, although this did not reach statistical significance.
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Analyzed as a whole, the 6 patients who were placed on cardiopulmonary bypass had elevations in IL-6, IL-8, and IL-10 when compared with patients not requiring bypass (all p < 0.01). However, the 4 bypass patients with PGF had higher IL-6 concentrations than the bypass patients with PLTRE and no injury (Fig 5).
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and IL-10 concentrations in the samples that had been exposed to the allograft (systemic arterial, p < 0.05). This is demonstrated most clearly for IL-10 in Figure 4, where systemic arterial concentrations of IL-10 are higher than pulmonary arterial samples (TNF not shown). This was not observed in the patients with PLTRE or in those without injury; and was not seen with IL-6 or IL-8. |
Comment |
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Clinical risk factors for primary graft failure in recipients have been previously identified. In a multivariate analysis of 225 patients, Christie and associates [2] found younger age (21 to 25 years old), older age (more than 45 years), and a diagnosis of primary pulmonary hypertension as risk factors for PGF. Our 4 PGF patients had significantly younger mean age than the other two injury groups; and PGF did develop in the only patient in this series with primary pulmonary hypertension.
Lung allograft ischemia time as a risk factor for PGF has been debated in the literature [2, 12, 15]. In the present study, longer ischemia times were observed in the patients with reperfusion injury, but they were not statistically distinct from those without injury, nor could a difference be observed within the two injury subgroups. Thabut and associates [12] had identified prolonged ischemia time as a risk factor in their PGF population, but their reported mean ischemia time of 282 ± 111 minutes was less than the mean of our severe injury cohort.
Our institution had previously characterized the inflammatory cytokine cascade after visceral ischemia-reperfusion injury in thoracoabdominal aortic aneurysm repair, and found elevations in TNF (> 150 pg/mL) and IL-6 (> 1,000 pg/mL) were associated with postoperative multiorgan dysfunction [6]. The goals of the current study were to examine the relationship between proinflammatory cytokine levels in the acute phase of lung allograft reperfusion and the severity of pulmonary allograft injury and to determine the source of these cytokines. Mal and colleagues [7] had examined cytokines on postoperative days 1 through 7, although this did not reflect the early response. However, Pham and colleagues [9] found early elevations in IL-6 did correlate with later allograft dysfunction.
In this study, only minimal concentrations of TNF were identified, which may have been a reflection of the immediate and 4-hour collection times that were based on our previous study with aneurysm repair [6]. Others have shown that TNF peaks as early as at 45 to 60 minutes, suggesting future studies will require earlier sampling times [16]. However, a significant difference in the TNF concentrations pre-lung and post-lung circulation was noted in the PGF patients, implicating the lung allograft or its resident macrophages in the production of this proinflammatory cytokine.
The most significant findings of this study were that patients with PGF (severe injury) exhibited graded increases in IL-6, IL-8, and IL-10 levels over PLTRE (moderate injury). The IL-6 concentrations peaked at 4 hours after reperfusion, and PGF patients displayed elevations of this cytokine over the PLTRE and no injury groups, respectively. The peak concentrations of IL-6 and the association with severity of injury further substantiate the study of Pham and coworkers [9] of IL-6 in lung transplantation. We were unable to identify the lung allograft as the source of IL-6, suggesting that other tissues rich in epithelial cells may have a role in its release during ischemia-reperfusion injury. Wen and associates [17] reported that pulmonary complications after liver transplantation were associated with the release of IL-6 from hepatocytes owing to an ischemia-reperfusion mechanism, suggesting that secondary lung injury may be directed through an IL-6 mechanism. Interleukin-6 is also implicated in T-cell stimulation and the generation of T-regulatory cells, both of which have been shown to play a role in the development of reperfusion injury in an animal model of lung transplantation [18–20].
Elevations of IL-8 in PLTRE have been previously established [8, 21, 22]. Increased IL-8 levels in both donor bronchoalveolar fluid and allograft tissue have been associated with primary graft failure, supporting the theory that IL-8 contributes to lung injury through increased chemotaxis and neutrophil recruitment [8]. We observed peak IL-8 concentrations at 4 hours after reperfusion, with persistent elevation through 24 hours, but did not find significant differences in IL-8 levels between the pre-lung and post-lung circulation, suggesting that the source of IL-8 is not likely the allograft.
The anti-inflammatory cytokine, IL-10, peaked at the time of reperfusion. One can speculate that the early release may be intended to limit the magnitude of the inflammatory response and tissue damage during the ischemic period, and to limit injury during reperfusion; this has been suggested in a thoracoabdominal aneurysm repair model at our institution [6, 23, 24]. Higher systemic arterial levels of IL-10 compared with pulmonary arterial levels suggest the lung allograft is one source of IL-10. Because IL-10 can mediate proinflammatory suppression both locally and systemically, it has been suggested as a method for limiting reperfusion injury. Several animal models have employed gene transfer of IL-10 to limit reperfusion injury, with encouraging results [25–27].
Cardiopulmonary bypass has been used to facilitate lung transplantation in bilateral recipients and in patients with severe pulmonary hypertension. Cardiopulmonary bypass has been identified as a risk factor for allograft dysfunction by Gammie and coworkers [28], although Christie and associates [2] were unable to identify cardiopulmonary bypass or bypass time as risk factors for PGF. Cardiopulmonary bypass has also been associated with an elevation in the proinflammatory cytokines [29]. In the present study, PGF developed in 4 of the 6 patients placed on bypass; but as shown in Figure 5, the patients in whom PGF developed had higher levels of IL-6, implicating IL-6 and not cardiopulmonary bypass per se in the etiology of PGF.
In summary, the present study found elevations in plasma IL-6, IL-8, and IL-10 were correlated with progressive injury in the recipient allograft after lung transplantation, especially IL-6. Elevations in systemic arterial concentrations of TNF and IL-10, as compared with pulmonary arterial samples, implicate the lung allograft as the origin of TNF and IL-10. Improved understanding of the cytokine cascade will enable the development of novel treatment strategies for the prevention of PLTRE and PGF.
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Discussion |
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One of the things that I took away from the presentation is that the only donor characteristic that you looked at was ischemic time. Additionally, your definition of injury looks at an A-a gradient of just under 300. In fact, as we try to expand our donor pool and transplant more patients, we're routinely looking at donors with A-a gradients between 200 and 300 and actually getting good results. So we may be shooting ourselves in the foot by using a definition where we're already starting out with a donor organ that by your criteria would be considered to have significant lung injury. I wonder if you could talk a little bit about the donors in this group.
I was also struck by the number of retransplants in your series. I think there were 6 retransplantations in this group of 22 patients. Retransplantation of the lung is a controversial ethical issue to begin with coupled with a very complex immunologic factor all thrown into the same mix. I wonder whether the study could be cleaner if you looked at more primary transplants and excluded those undergoing retransplantation.
DR MATHUR: Thank you for your comments and your excellent questions. In response to donor factors, we looked at graft ischemia time as our primary donor criteria. The experience with graft ischemia times in terms of its contributions to reperfusion injury has been met with mixed results. In animal models, graft ischemia time has been shown in several models of rat lung transplantation to be an important indicator for subsequent injury, but in clinical transplantation the graft ischemia has been met with mixed results. Certainly in our experience we did not see a statistically significant difference, but we did see longer graft ischemia times.
In response to your question about using the A-a gradients, the A-a gradients that we used were in order to define clinical injury after transplantation. In terms of looking at donor characteristics before transplantation, our study did not include looking at those clinical criteria.
DR DECAMP: Based on your data, would you recommend pretreating the donor or pretreating the recipient with anything to abrogate some of this cytokine release?
DR MATHUR: Well, our study is a descriptive study, and we were able to show an association with cytokine proliferation with subsequent reperfusion injury. There has been attention to animal studies looking at the gene transfer of IL-10 to a transplant recipient. These models have been met with really good results, they have shown less reperfusion injury, and certainly that is a potential study for the future.
DR DAVID A. FULLERTON (Denver, CO): Amit, I enjoyed your study very much and I compliment you on your presentation and for the fact that, as a medical student, you're taking great advantage of Dr Beaver's laboratory. So it's a job well done.
The comments I have about your study are two things. One, the rise in IL-10, as an anti-inflammatory cytokine, one would hope it could be therapeutically manipulated to suppress the production of proinflammatory cytokines, and I was curious as to whether or not you may have some insight as to how one might do that.
Second, most of the patients like this in our own institution would be immediately started on inhaled nitric oxide, and I was curious if any of your patients were, and, if so, if the use of inhaled nitric oxide might have changed the cytokine profiles that you identified.
DR MATHUR: Doctor Fullerton, I thank you for your kind comments. With regard to your first question about the use of IL-10 and its manipulation for therapeutic intervention, certainly animal models have shown promising results. The WashU group, Itano and colleagues in the Patterson laboratory, have shown that transfection of IL-10 in a rat model was able to mitigate reperfusion injury. The anti-inflammatory effects of IL-10 in vitro have been shown in many models and certainly would be a potential place for therapeutic intervention. In response to your question about nitric oxide, I cannot comment specifically on which patients in this study received nitric oxide, but it certainly is something that is used in other institutions. Depending on the time that it's begun, it could have a potential role in mitigating the late peaks in cytokine release.
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Acknowledgments |
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References |
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This article has been cited by other articles:
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  S. A. Daud, R. D. Yusen, B. F. Meyers, M. M. Chakinala, M. J. Walter, A. A. Aloush, G. A. Patterson, E. P. Trulock, and R. R. Hachem Impact of Immediate Primary Lung Allograft Dysfunction on Bronchiolitis Obliterans Syndrome Am. J. Respir. Crit. Care Med., March 1, 2007; 175(5): 507 - 513. [Abstract] [Full Text] [PDF]
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