Brain death-induced cytokine release is not associated with primary graft dysfunction: a cohort study

Objective To examine the association between donor plasma cytokine levels and the development of primary graft dysfunction of organs transplanted from deceased donors. Methods Seventeen deceased donors and the respective 47 transplant recipients were prospectively included in the study. Recipients were divided into two groups: group 1, patients who developed primary graft dysfunction; and group 2, patients who did not develop primary graft dysfunction. Donor plasma levels of TNF, IL-6, IL-1β, and IFN-γ assessed by ELISA were compared between groups. Results Sixty-nine organs were retrieved, and 48 transplants were performed. Donor plasma cytokine levels did not differ between groups (in pg/mL): TNF, group 1: 10.8 (4.3 - 30.8) versus group 2: 8.7 (4.1 - 33.1), p = 0.63; IL-6, group 1: 1617.8 (106.7 - 5361.7) versus group 2: 922.9 (161.7 - 5361.7), p = 0.56; IL-1β, group 1: 0.1 (0.1 - 126.1) versus group 2: 0.1 (0.1 - 243.6), p = 0.60; and IFN-γ, group 1: 0.03 (0.02 - 0.2) versus group 2: 0.03 (0.02 - 0.1), p = 0.93). Similar findings were obtained when kidney transplants were analyzed separately. Conclusion In this sample of transplant recipients, deceased donor plasma cytokines TNF, IL-6, IL-1β, and IFN-γ were not associated with the development of primary graft dysfunction.

inflammatory infiltrate in the glomerular tubules of brain-dead animals. (9) In line with this finding, Contreras et al. demonstrated that BD-induced inflammatory activity had an adverse impact on islet function in rats, increasing apoptosis in beta cells. (10) Primary graft dysfunction is a common complication of deceased donor organ transplantation. This dysfunction is associated with increased risk of graft loss in the first 36 months of follow-up, as well with increased length of hospital stay (11) and increased costs. (12) The association between BD and PGD, however, is not fully understood. It is supposed that by activating the inflammatory cascade, BD can be a key component of ischemia-reperfusion injury, an effect that might be even more pronounced in organs from expanded criteria donors. Interestingly, there is an association for the development of PGD across different organs transplanted from the same donor. (13) The present study was designed to examine the association between donor plasma tumour necrosis factor (TNF), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interferon-gamma (IFN-γ) and the development of PGD of organs transplanted from deceased donors.

Deceased donors and transplant recipients
The study protocol was approved by the Ethics Committee (number 13-060) at Hospital de Clínicas de Porto Alegre, in the city of Porto Alegre, and three other participating transplant centers: Hospital São Lucas and Hospital Dom Vicente Scherer, both in Porto Alegre, and Hospital São Vicente de Paulo, in Passo Fundo, in agreement with the Helsinki declaration of 1975. All institutions are in Rio Grande do Sul, the southernmost state of Brazil. Informed consent was obtained from the patients' legal representatives. Brain death was assessed independently by two physicians and was based on the following criteria: coma with complete unresponsiveness, absence of brain stem reflexes, apnea test, and confirmatory test with absence of cerebral blood flow according to the Brazilian law. (14) From November 2010 to December 2011, brain-dead patients older than 18 years admitted to the intensive care unit at Hospital de Clínicas de Porto Alegre were prospectively included in the study after the first clinical examination consistent with BD. Blood samples were collected at study entry. Organ recipients were identified from a crossover list provided by the regional organ distribution center. Clinical and laboratory data were recorded for brain-dead donors and transplant recipients.

Statistical analysis
Categorical variables were expressed as percentages. Quantitative data were expressed as the mean and standard deviation (SD) if normally distributed. Variables with skewed distributions were log-transformed before analysis and expressed as median and minimum-maximum. Groups were compared using Student's t test, chi-square test or Fisher's exact test, as appropriate. Spearman's rank correlation was used to assess correlations between different quantitative variables. A sample size of 32 organ transplant recipients (16 patients with PGD and 16 patients without PGD) was required to detect a difference of at least one SD in TNF log, (4) considering a power of 80% and an alpha-error of 5%. Values were considered to be statistically significant if p < 0.05. All statistical analyses were performed using Statistical Package for Social Science (SPSS), version 18.0 (Chicago, IL).

RESULTS
A total of 69 organs were retrieved from 17 deceased donors (a mean of 4.05 organs per donor): 34 kidneys, 17 pancreases, 13 livers, four lungs, and one heart. All pancreases were used for research purposes, and one liver and two lungs were discarded due to technical problems. Forty-eight transplants were performed in 47 patients (one patient underwent a simultaneous liver-kidney transplant). The characteristics of 38 transplant recipients (data were not available for nine patients) and 17 deceased donors included in the study are summarized in table 1. Briefly, 70% of donors were men with a mean ± SD age of 54 ± 11 years; stroke was the leading cause of BD (76.5%) followed by anoxic encephalopathy (23.5%). All deceased donors required vasopressor support, and sepsis was present in 41%.
In a logistic regression model with PGD as the dependent factor and donor age, duration of ventilatory support, cold ischemia time, TNF, and IL-6 as cofactors, cold ischemia time was the only variable associated with PGD development (odds ratio -OR = 0.85, 95% confidence interval -95%CI 0.74 -0.98, p = 0.032).

DISCUSSION
In this sample of recipients of organs from deceased donors, BD-induced plasma cytokine release was not associated with PGD, as donor plasma TNF, IL-6, IL-1β, and IFN-γ levels did not differ between transplant recipients who developed PGD and those who did not.
Primary graft dysfunction is a serious complication of transplantation that results from ischemia-reperfusion injury, a process triggered by the systemic inflammatory state of the donor. (20) Patients who died within 30 days of lung transplant had elevated levels of IL-6 in biopsies prior to implantation. (21) Likewise, myocardial TNF mRNA expression during organ retrieval predicted right ventricular dysfunction in heart recipients. (8) Moreover, liver biopsies from deceased donors showed higher CD4 and CD8 infiltration than biopsies from living donors. (22) Taken together, these studies suggest an association between increased organ tissue inflammation and PGD. However, our study did not show an association between plasma cytokine levels and PGD. One possible explanation for the lack of association between systemic cytokine levels and PGD, in contrast with the findings reported for tissue levels, is that the degree of tissue inflammation is higher than that measured in plasma, suggesting that tissue inflammation levels might be a better predictor of PGD than plasma inflammation levels. Furthermore, the high rate of DGF in kidney recipients (60.7%) observed in our sample of patients, which is consistent with that reported in another Brazilian study, (11) might have prevented us from detecting a difference between groups.
Murugan et al. recruited 30 deceased donors and analyzed the outcomes of the respective 78 transplant recipients. In this cohort of patients, higher plasma IL-6 levels but not TNF and IL-10 levels before organ procurement correlated with a trend to lower 6-month hospital-free survival in recipients. (19) Therefore, we used the cut-off value of IL-6 suggested in their study (193pg/ mL), but no association with PGD was found for values above or below this threshold, suggesting that plasma IL-6 may not be a good predictor of early outcomes, such as PGD. In the short term, cold ischemia time and donor age appeared to be better predictors of outcome, as demonstrated previously (11,23,24) and supported by our findings.
In a previous study, we demonstrated an upregulation of plasma TNF and IL-6 in deceased donors compared to controls. (4) Interestingly, in the study by Murugan et al., plasma concentrations of IL-6 immediately before organ procurement were lower in donors treated with corticosteroids than in untreated donors. (19) Likewise, Kotsch et al., in a randomized controlled trial, showed that methylprednisolone therapy in deceased donors reduces inflammation in the donor liver and improves outcome after liver transplantation. (25) However, we did not find an association between plasma cytokine levels and liver PGD.
Elevated plasma IL-6 levels have been associated with a poorer prognosis in a variety of critical care settings (26)(27)(28) and with lower organ yield in transplantation settings. (19) However, to the best of our knowledge, this study is the first to evaluate donor plasma TNF, IL-6, IL-1β, and IFN-γ levels as predictors of PGD development in organs transplanted from brain-dead donors. This study had several limitations. First, the sample size was calculated to detect a difference of one SD in TNF log between brain-dead and control patients without brain-dead in a previous study (4) and, in fact, may be underpowered to detect differences in PDG development between groups of transplant recipients. However, when estimating the sample size required to detect a difference in the present study, a sample size of at least 770 organ transplant recipients would be necessary. Second, we measured plasma cytokines only at the time of organ procurement, which may have been late in the inflammatory process, as cytokines peak earlier after BD. Third, we believe that a time course with earlier time points, such as 1, 2 and 6 hours, and up to 12 hours, might provide more consistent information about inflammation in brain-dead donors and its association with PGD development.

CONCLUSION
Primary graft dysfunction is a predictor of worse shortand long-term outcomes after transplantation. In this respect, detecting clinical and laboratory variables that can accurately predict the development of primary graft dysfunction would be clinically relevant. Plasma cytokines can be easily and quickly measured, but the small sample size and the single time point measurement of plasma cytokines in our study preclude a conclusion regarding the association of donor plasma TNF, IL-6, IL-1β, and IFN-γ values with development of primary graft dysfunction. Then, the role of inflammatory cytokines, as a possible pathway associated with primary graft dysfunction development, should be the focus of investigation of larger studies.

Authors' contributions
TH Rech participated in study conception and design, data acquisition, analysis and interpretation of data, statistical analysis, drafting and revision of the manuscript. G Custódio, LV Kroth, S Henrich, and EM Rodrigues Filho participated in data acquisition. D Crispim participated in study conception and revised the manuscript. CB Leitão participated in study conception and design, interpretation of data, statistical analysis and revised the manuscript. TH Rech is the guarantor of this work and, as such, has full access to all data and takes responsibility for the integrity of the data and the accuracy of the data analyses. We thank the regional organ distribution center for providing the donor-recipient crossover list.