IMMUNE STATUS AND APOPTOSIS ACTIVATION DURING BRAIN DEATH
Adrie, Christophe*†; Monchi, Mehran‡§; Fulgencio, Jean-Pierre∥; Cottias, Pascal¶; Haouache, Hakim*; Alvarez-Gonzalvez, Antonio*; Guerrini, Patrice**; Cavaillon, Jean-Marc††; Adib-Conquy, Minou††
*Intensive Care Unit, Delafontaine Hospital, Saint Denis; †Department of Physiology, Cochin Hospital, Paris Descartes University, AP-HP, Paris; ‡Intensive Care Unit, Jacques Cartier Institute, Massy, France; §Intensive Care Unit, Liège University Hospital, Liège, Belgium; ∥Intensive Care Unit, Tenon Hospital, AP-HP, Paris; ¶Surgical unit, Victor Dupouy's Hospital, Argenteuil; **Biomedicine Agency, Kremlin-Bicêtre Hospital, Le Kremlin Bicêtre; and ††Institut Pasteur, Unit Cytokines & Inflammation, Paris, France
Received 10 Jun 2009; first review completed 23 Jun 2009; accepted in final form 25 Jun 2009
Address reprint requests to Jean Marc Cavaillon, M.D., Dr. Sc., Unit Cytokines & Inflammation, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail: email@example.com.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journalss Web site (http://www.shockjournal.com/).
Conflict of Interest: Christophe Adrie received a grant from the publicly funded Biomedicine Agency, which manages organ donor data in France.
The present study evaluates the role of the inflammatory status and apoptosis activation in the development of organ dysfunction after brain death using plasma assays and macroarray analysis on skeletal muscle biopsies to look for evidence of remote tissue damage in two intensive care units in France and one in Belgium. As controls, we used patients undergoing hip surgery and healthy volunteers. Causes of brain death in the 85 consecutive patients included in the study were cardiac arrest (n = 29; 34%), stroke (n = 42; 49%, with 38 patients having hemorrhagic stroke), and head injury (n = 14; 17%). Of the 85 patients, 45 donated 117 organs. Plasma endotoxin and cytokine levels indicated a marked systemic inflammatory response in brain-dead patients, which was strongest in the cardiac arrest group. Leukocyte dysfunction, as assessed by cytokines production in response to various stimuli, was noted in a subgroup of patients with brain death after stroke. Interestingly, skeletal muscle biopsies showed no increase in mRNAs for genes related to inflammation, whereas mRNAs for both antiapoptotic and proapoptotic genes were increased, the balance being in favor of apoptosis induction. The increased activation of the proapoptotic caspase 9 was further confirmed by Western blot. In conclusion, the presence of inflammation and apoptosis induction may explain the rapid organ dysfunction seen after brain death. Both abnormalities may play a role in organ dysfunction associated with brain death. However, the level of systemic inflammation or the presence of circulating endotoxin was not associated with lower graft survival.
Brain death is defined as irreversible damage to the brain stem combined with lesions of the cerebral hemispheres and constitutes the legal definition of death required for organ donation (1). The central insult, whether secondary to a catastrophic increase in intracranial pressure or more gradual in onset, produces profound physiologic and structural derangements in the peripheral organs (2). Brain death influences allograft survival and affects the time-course and severity of early graft rejection. This fact is illustrated not only by the similar survival rates of allografts from living unrelated donors and from haplotype-matched living related donors but also by the consistently better results obtained with organs from living donors compared with cadavers (3). There is a consensus that specific treatments are in order to improve the quality of organs from brain-dead donors before transplantation, with the goal of improving early and long-term graft function after transplantation (4). To determine which treatments are most effective, we need to understand the pathophysiologic mechanisms involved in post-brain death organ damage. A recent study (5) showed significant up-regulation of various cytokines in brain-dead donors compared with living donors, as well as greater severity of I/R injury after transplantation of livers from brain-dead donors. Inflammation may be at least partly responsible for the hormonal and acute-phase-reactant abnormalities that occur after brain death (6) and may increase the risk of acute graft failure (7-9).
We hypothesized that brain death is associated with an early systemic inflammatory response, possibly combined with activation of apoptotic cell death, two events that may contribute to induce rapid organ dysfunction. We studied the immunological profile after brain death and examined skeletal muscle biopsies to look for evidence of inflammation and increased apoptosis in peripheral tissue.
PATIENTS AND METHODS
Brain-dead patients and controls
This study was approved by the ethics committee (Pitié-Salpêtrière Hospital, Paris, France), and informed consent was obtained from the next of kin of each patient. All consecutive brain-dead patients older than 18 years were prospectively included (Delafontaine, Tenon, and Liege Hospitals). Both the Simplified Acute Physiology Score (10) and the Sepsis-related Organ Failure Assessment (SOFA) score (11) were calculated during the 24 h after admission. Brain death was assessed independently by two physicians according to French law based on the following criteria: coma with complete unresponsiveness, including absence of all brain stem reflexes confirmed using an apnea test; two isoelectric electroencephalograms, recorded over at least 30 min at an interval of at least 4 h (core temperature greater than 35°C; absence of drug poisoning); or absence of cerebral blood flow. Patients were categorized into three groups based on whether the cause of brain death was cardiac arrest (anoxia), head injury, or stroke. None of the patients received thyroxine or methylprednisolone.
Information on all transplant donors and recipients in France is entered prospectively into the database (CRISTAL) of the Biomedicine Agency (nationwide organ transplant agency). Delayed renal graft function was defined as a need for dialysis within 1 week after transplantation, followed by recovery of renal function.
The controls were eight healthy volunteers and nine patients undergoing hip surgery for degenerative noninflammatory hip disease.
Blood collection (endotoxin-free tubes; Becton Dickinson, Franklin Lakes, NJ) was performed at the diagnosis of brain death and just before organ procurement along with muscle biopsies done in the vastus lateralis thigh muscle via a small incision and frozen immediately in liquid nitrogen.
Plasma cytokine assays using BioPlex
The following cytokines and chemokines were assayed: IL-6, IL-8, IL-1Ra, IL-10, TNF, chemokine (C-C motif) ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) using the BioPlex system (BioRad, Hercules, Calif).
Cytokines and soluble-factor detection by Enzyme-Linked Immunosorbent Assay
We used specific enzyme-linked immunosorbent assay kits to assay plasma levels of sFas, sFasL, and sTRAIL (DuoSet; R&D Systems, Minneapolis, Minn). TNF, IL-10, and IFN-γ levels were measured in whole-blood culture supernatants using specific enzyme-linked immunosorbent assay kits (TNF and IL-10: DuoSet, R&D Systems; IFN-γ: OptEIA, BD Biosciences).
Whole blood was cultured in 24-well plates as already described (see electronic supplement). Because leukocyte dysfunction is known to occur after successfully resuscitated cardiac arrest (12), we confined these tests to patients with stroke, among whom we excluded patients with evidence of infection.
The samples were diluted in LAL reagent water (Biowhittaker) in nonpyrogenic 96-well culture plates using nonpyrogenic pipettes and tips (Costar). Bacterial endotoxin was detected using a diazo-coupling LAL assay (Associates of Cape Cod, East Falmouth, Mass). Optical density was read at 570 nm, and the detection limit was 0.02 endotoxin units (EU) mL−1 (for more details, see online supplement).
RNA preparation from skeletal muscle biopsies
RNA was extracted from the biopsies using Trizol reagent (1 mL Trizol per 200 mg of tissue; Invitrogen, Carlsbad, Calif). The RNA was further purified using the RNeasy kit (Qiagen, Courtaboeuf, France). RNA integrity was checked on agarose gel containing ethidium bromide, and RNA concentration was determined by measuring the optical density at 260 nm.
Synthesis, labeling of cRNA, and macroarray experiments
We synthesized cRNA using 3 μg of total RNA and the True Labeling-AMP 2.0 kit (SuperArray, Frederick, Md). The cRNA was labeled by incorporation of biotin-UTP (Perkin Elmer, Boston, Mass) then purified using the cRNA cleanup kit (SuperArray). We used 2 μg of cRNA for hybridization with Oligo GEArray DNA macroarrays (OHS-012 and OHS-803, SuperArray; for more details, see online supplement). After hybridization, the spots corresponding to each gene were quantified using a charge-coupled device camera system (ChemiDoc XRS, Biorad) and Quantity One 4.6.1 software (BioRad); the background was subtracted, and values were adjusted based on the signal intensity of housekeeping genes (ribosomal RNA, GAPDH, β2-microglobulin).
Caspase 9 Western blot
Proteins were extracted from skeletal muscle biopsies using lysis buffer containing inhibitors of proteases and phosphatases. Protein extracts (3 mg) from each patient were immunoprecipitated with an anti-caspase 9 antibody detecting both the proform and the cleaved form (Cell Signaling Technology, Danvers, Mass; for more details, see the online supplement). The immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose sheets (Hybond C, GE Healthcare, Piscataway, NJ). Western blot was performed using the same anti-caspase 9 antibody and a secondary anti-rabbit antibody coupled to horseradish peroxidase (TrueBlot). Peroxidase activity was detected using the Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, Ill) and a charge-coupled device camera (ChemiDoc XRS, BioRad). The bands corresponding to caspase 9 were quantified using Quantity One 4.6.1 software (BioRad).
Data were expressed as median (interquartile range) or mean ± SEM, as appropriate. The value zero was assigned to values less than the assay detection limit. Within- and between-group differences were analyzed using Wilcoxon signed-rank tests, Mann-Whitney U tests, Kruskal-Wallis tests, or chi-square tests. The relationships between two continuous variables were evaluated using Spearman rank correlation tests.
To evaluate associations between cytokines and graft survival, cytokine levels were converted to categorical ordered variables by considering the quartiles of the baseline concentrations. Cox regression analysis was used to evaluate the association between each cytokine at baseline (expressed as ordered quartiles) and graft survival. We tested the proportionality of each cytokine using single-variable Cox analysis with Schoenfeld residuals; P < 0.10 was taken to indicate absence of proportionality.
P < 0.05 was considered significant. Statistical analyses were performed using Stata 9.2 software (Stata Corp., College Station, Tex).
A multiple-test procedure (multproc command in stata 9) calculated a corrected overall critical P value, which has the feature that an individual null hypothesis is considered to be acceptable if and only if its corresponding P value is greater than the corrected overall critical P value (13).
From February 1999 to April 2006, we included 85 consecutive patients. The cause of brain death was cardiac arrest in 29 patients (including 2 cardiac arrest secondary to brain hemorrhage), stroke in 42 patients (38 [90%] with hemorrhagic stroke), and head injury in 14 patients. Cardiac arrest patients had significantly worse organ dysfunction as assessed by the admission SOFA score. However, all 85 patients required large amounts of fluids, and 80 required vasopressor support, without significant differences across causes of brain death (Table 1). Of the 8 patients with documented infection, 7 had pulmonary infections, and 1 had a urinary tract infection.
One hundred and seventeen collected organs from 45 patients were subsequently transplanted (69 kidneys, 35 livers, 6 hearts, 4 lungs, and 3 pancreases). Four kidneys collected from 2 patients were found to be unsuitable for transplantation. Median time from kidney transplantation to last renal replacement therapy (if any) was 2.5 days (0-10), with no differences according to cause of death. Overall 1- and 5-year graft survival rates were 97% and 74% for kidneys, 100% and 61% for livers, and 100% and 100% for hearts, respectively. We also studied eight healthy controls (median age, 47 years [41-53]; 62% male) and patients undergoing hip surgery for degenerative disease (median age, 53 years [46-66]; 55% male).
Plasma cytokines, soluble factors, and endotoxin
Plasma cytokines were elevated in all three cause-of-death groups and more pronounced in the cardiac arrest group with the exception of TNF-α (Table 2). Among the chemokines, IL-8 and MCP-1 were higher in brain-dead patients than in healthy controls, whereas RANTES and IP-10 were similar. Although soluble Fas levels were elevated in brain-dead patients, levels of its ligand and of another proapoptotic molecule, TNF-related apoptosis-inducing ligand (TRAIL), were decreased, compared with healthy controls. The results of repeat assays performed in those patients who donated organs were not significantly different from the levels at diagnosis of brain death and at organ donation (see Figure, Supplemental Digital Content 1, illustrating plasma levels of cytokines and endotoxin at diagnosis of brain death (T1) (n = 45) and before organ donation (T2) (n = 45), with values of healthy controls (n = 8) and hip surgery (n = 9) provided for comparison, http://links.lww.com/SHK/A20), nor did the presence of infection affect the assay results (data not shown). Circulating endotoxin was detected in the plasma of 55% of cardiac arrest patients, 24% of stroke patients, and 46% of head trauma patients (Fig. 1). No endotoxin was detected in control groups. Levels of cytokines, soluble factors, or the presence of circulating endotoxin were not associated with graft survival all pooled (Fig. 2; for kidneys).
Production of cytokines in whole-blood assays in response to in vitro stimulation
Basal production of TNF-α and IL-10 was low or undetectable in patients (n = 8) and healthy volunteers (n = 14; Fig. 3, A and B). Leukocyte TNF-α production after LPS exposure was impaired in patients with brain death due to stroke, compared with healthy volunteers, and was unchanged after stimulation by heat-killed Staphylococcus aureus (SAC) or Escherichia coli. Conversely, patient leukocytes were more responsive to these three agents, producing significantly larger amounts of IL-10 than leukocytes from healthy controls. The ability of the leukocytes to produce large amounts of IL-10 and unaltered amounts of TNF-α in response to E. coli or SAC demonstrates that the cellular machinery remains fully functional.
We also tested the ability of T lymphocytes from brain-dead patients to produce cytokines in response to Concanavalin A (ConA) and phytohemagglutinin (PHA). ConA-induced IFN-γ production was considerably less marked with T lymphocytes from brain-dead patients compared with healthy volunteers (Fig. 3, C and D). IL-10 production in response to PHA and ConA was similar in the two groups. This last finding demonstrates that alterations in T-cell reactivity also vary across triggering agents and cytokines.
Evaluation of inflammation in skeletal muscle biopsies from brain-dead patients
The high plasma levels in brain-dead patients of several cytokines associated with inflammation, as well as of chemokines and endotoxin, prompted us to look for evidence of inflammation within tissues. The mRNAs were measured in skeletal muscle biopsies from 18 brain-dead patients (6 per group) and from 6 hip-surgery patients by macroarray, allowing the analysis of the expression of 440 genes. The degree of variability of the housekeeping genes from one chip to another was quite low. The integrated volumes were of (median and interquartile): 15945 , 16420 , and 4318  for ribosomal protein, GAPDH, and B2M, respectively. The inflammatory response was minimal in biopsies from brain-dead patients, with few genes being expressed and no significant differences with hip-surgery patients (Table 3). We found low levels of expression of molecules involved in cell signaling, cytokines, growth factors, one chaperone, and one proapoptotic protein. Although myocytes can share similar gene expression with leukocytes, particularly for cytokine expression (14), we cannot exclude, particularly for low values, that some gene expression may reflect the presence of mononuclear cells. Only four genes showed different levels of expression between brain-dead and hip-surgery patients. SERPINA3, TNF receptor II (TNFRII or TNFRSF1B), and lactate dehydrogenase mRNA expressions were significantly increased after brain death, whereas IL-26 mRNA showed a trend toward decreased expression (Fig. 4).
Apoptosis in skeletal muscle biopsies from brain-dead patients
In addition to plasma apoptosis markers (sFas, sFasL, and TRAIL), we measured the expression of mRNAs coding for 112 genes involved in apoptosis. Again, we used macroarray technology to examine skeletal muscle biopsies from 18 brain-dead patients (6 per group) and from 6 hip-surgery patients. The genes fell into three groups based on whether they were expressed at similar levels in brain-dead patients and in controls, expressed at significantly higher levels in brain-dead patients, or only expressed in brain-dead patients.
Genes showing no significant differences in expression between the two groups (Table 4) consisted of both proapoptotic genes, including Bcl-2 antagonist/killer1, Bcl-2 like 13, Bad, Bax, BCL2/adenovirus E1B 19 kDa interacting protein 3, and apoptotic protease activating factor 1; and of antiapoptotic genes, including Bcl-2 like 12, Bcl-2-associated athanogene (BAG) 4, TRAIL decoy receptor 1, and nucleolar protein 3. Detected mRNAs for signaling molecules included those coding for TNF receptor-associated factor 5, and caspase recruitment domain family member 14, both of which are involved in nuclear factor-κB activation. Finally, mRNA coding for growth arrest and DNA-damage-inducible protein α (Gadd45a), a gene involved in DNA repair, was also detected in some of the biopsies from brain-dead patients. Although this gene was not expressed in any of the hip-surgery patients, it was not expressed by all brain-dead patients, and consequently, the difference between the two groups was not statistically significant.
Figure 5 shows the genes whose expression differed significantly between brain-dead patients and hip-surgery patients. The left-hand panel shows the genes that were expressed in both groups at higher levels in the brain-dead patients than in the hip-surgery patients. This group includes myeloid cell leukemia sequence 1 (MCL-1), BAG1 and BAG3, and cellular inhibitor of apoptosis 1 (cIAP1, also known as baculoviral IAP repeat-containing 2). All these genes were shown to inhibit apoptosis. In contrast, the genes shown in the right-hand panel, which were expressed only in the brain-dead patients, encoded proapoptotic factors. This group consists of caspase 9, caspase 14, BCL2/adenovirus E1B 19 kDa interacting protein (BNIP) 2, and DNA fragmentation factor 40 kDa (DFF40).
Caspase 9 contributes to cell death through the intrinsic apoptosis pathway by activating caspase 3, thus enabling activation of the enzyme DFF40. However, increased expression of caspase 9 mRNA is not sufficient to increase caspase 9 activity because caspase 9 is produced as a proform that must be cleaved to become active. Therefore, we used protein extracts from skeletal muscle biopsies of 11 other brain-dead patients to measure caspase 9 expression using immunoprecipitation, followed by Western blotting. We used an anti-caspase 9 antibody that detects both the pro-caspase 9 and its active cleaved form. As shown in Figure 6A, the cleaved form was readily detectable in brain-dead patients, whereas its levels were low or undetectable in hip-surgery patients. Quantification of the band corresponding to caspase 9 showed a significantly higher expression in brain-dead patients (Fig. 6B).
A systemic inflammatory response was documented in all brain-dead patients irrespective of the cause of death but more pronounced after cardiac arrest. Plasma endotoxin and leukocyte dysregulation were also observed. In muscle biopsies, expression of inflammatory cytokine mRNAs was low, whereas expression for proapoptotic proteins was increased. Most importantly, graft survival was neither associated with the inflammatory response as assessed by cytokine assays nor with circulating endotoxin levels.
A systemic inflammatory response was consistently present at the diagnosis of brain death, as shown by the levels of plasmatic proinflammatory and anti-inflammatory cytokines. Not surprisingly, inflammation and organ dysfunction as assessed by the SOFA score were more marked in the cardiac arrest group. Despite the higher systemic inflammatory response, the organs (kidneys, livers, or even heart) of these have been repeatedly shown to have a similar long-term outcome than those obtained from patients who died from others causes (15-17).
As expected, many markers known to be elevated in the systemic inflammatory response syndrome were high in brain-dead patients. Only RANTES was not significantly different from healthy controls, in contrast to the previously reported decrease in septic patients (18). Surprisingly, IP-10, a chemokine induced by IFN-γ, was not different compared with controls, in apparent contradiction to the up-regulation of IP-10 mRNA reported in kidneys from brain-dead patients (19) and to the induction of IP-10 by LPS injection in human volunteers (20).
Brain injury is primarily ascribed to I/R syndrome: initially, increased intracranial pressure and decreased cerebral blood flow lead to massive catecholamine release, which causes marked vasoconstriction, followed by hypotension, hemodynamic instability, and further exacerbation of the inflammatory response to reperfusion (21). Although I/R injury occurs in brain death due to all causes, it is most marked in successfully resuscitated cardiac arrest patients who experience whole-body I/R starting at the time of cardiac arrest.
Apoptosis and inflammation in patients with brain death may damage the organs collected for transplantation. We chose skeletal muscle as the biopsy site to include biopsies from hip-surgery patients as controls and therefore to correct for possible bias due to tissue sampling and handling. Surprisingly, whereas plasma levels of inflammatory cytokines were high, inflammation in muscle biopsies was minimal. This was in contrast with some animal models or studies in other clinical settings (14, 22, 23). Skeletal muscle may be less sensitive to inflammation compared with organs collected for transplantation. However, in a rat model of I/R, skeletal muscle showed progressive histological damage and inflammatory infiltrates (24). In our brain-dead patients, only three genes were significantly up-regulated: lactate dehydrogenase, SERPINA3, and TNFRSFI. Lactate dehydrogenase is a marker for muscle damage and hypoxia. SERPINA3, which was undetectable before hip surgery, is a serine proteinase inhibitor also known as α1-antichymotrypsin. The level of this acute-phase protein increases during acute and chronic inflammation. SERPINA3 neutralizes cathepsin G, which may be released by neutrophils upon activation (25). Thus, increased SERPINA3 expression may reflect neutrophil infiltration in tissues after brain death. Finally, the circulating levels of the soluble form of TNFRSF1 (also known as TNFRII) increase after severe inflammation or infection (26). Increased TNFRSF1 mRNA expression has been reported in rat skeletal muscle after endotoxin administration in vivo (27).
We found plasma endotoxin in 38% of our patients, suggesting that the systemic inflammatory response associated with brain death may induce gut damage with translocation of endotoxin, further promoting inflammation and remote organ injury. Endotoxinemia may promote the development of an endotoxin tolerance-like phenomenon in brain-dead patients (28). Fatal stroke was associated with dysregulated leukocyte response to LPS, as already described in patients after cardiac arrest (12). "Leukocyte reprogramming" aptly describes this phenomenon because the leukocytes remain responsive to whole heat-killed bacteria and produce large amounts of IL-10 in response to LPS (29). Reprogramming affects not only monocytes but also lymphocytes. Indeed, we found that the IFN-γ response to the T-cell mitogen ConA was diminished, whereas the IL-10 response was normal. The T-cell response thus varies with the nature of the activating signal (30).
The macroarray analysis showed that skeletal muscle from brain-dead patients expressed several genes involved in apoptosis. Among them, MCL-1, BAG1, BAG3, and cIAP1 were less expressed in biopsies from hip-surgery patients compared with brain-dead patients. Myeloid cell leukemia sequence 1 is an antiapoptotic member of the Bcl-2 family that regulates the mitochondrium-mediated pathway of apoptosis, maintains mitochondrial integrity, and its up-regulation modulates apoptosis in several cell types, including monocytes and neutrophils during inflammation and sepsis (31). The up-regulation of MCL-1 may be related to the increased levels of IL-6 observed in these patients (32, 33). Bcl-2-associated athanogene 1 and BAG3 (34), two other genes that were expressed at lower levels in hip surgery than in brain-dead patients, belong to a conserved family of cytoprotective proteins that bind to and regulate molecular chaperones of the heat shock protein 70 family. B Bcl-2-associated athanogene 3 was prominently expressed in skeletal muscle, and both Bag1−/− and Bag3−/− mice exhibit massive apoptosis (35, 36). Finally, increased expression of cIAP1 is associated with reduced apoptosis. Neutrophil stimulation by LPS in vitro induced the expression of cIAP1 mRNA (37, 38). This may contribute to explain the up-regulation of cIAP1 in brain-dead patients because circulating endotoxin was detected in 38% of these individuals. Expression of the antiapoptotic molecule Bcl-2 was not diminished in muscle biopsies from brain-dead patients, in contrast to results obtained with kidney biopsies from brain-dead patients (19).
Four proapoptotic genes were expressed in muscle biopsies from brain-dead patients but not in those from hip-surgery patients: BNIP2, caspase 14, caspase 9, and DFF40. Caspases are cysteine-dependent aspartate-specific proteases that are crucial for the initiation and execution of apoptosis. Some caspases such as caspase 1 are involved in the inflammatory response and are required for the maturation of IL-1β and IL-18. Caspase 14 expression is developmentally regulated and occurs chiefly in keratinocytes (39). However, caspase 14 expression has been reported at other sites in some diseases such as ulcerative colitis (40) or the brain in a canine model of cardiac arrest (41). Caspase 9, a cell-death initiator, is activated within the apoptosome complex after mitochondrial stress. Active caspase 9 may then interact with pro-caspase 3, inducing its cleavage and activation. In addition to up-regulation of caspase 9 mRNA after brain death, we identified the cleaved form of caspase 9 by Western blot. This cleaved form, similarly to caspase 9 mRNA, was absent in the biopsies from hip-surgery patients. The step leading to DNA fragmentation requires the enzyme DFF, which is found in the nucleus as a heterodimer formed by the inhibitory subunit DFF45 and the latent nuclease subunit DFF40. Upon apoptosis activation, DFF45 is cleaved and DFF40 released. We found that DFF40 mRNA was expressed in biopsies from brain-dead but not hip-surgery patients, suggesting an induction of apoptosis. Similarly, previous studies found evidence of apoptosis induction in various organs (19, 42-44), including the heart in rabbits (43) and humans, most notably those with heart dysfunction (42). A scheme of the apoptosis pathways that were differentially affected by brain death is available online (see Figure, Supplemental Digital Content 2, http://links.lww.com/SHK/A21). Elements with gradation of gray were similarly expressed in brain-dead patients and hip-surgery patients. Dark gray elements correspond to genes that were significantly up-regulated in brain-dead patients.
Interestingly, we found no association between cytokine or endotoxin levels and graft survival. In contrast, serum procalcitonin elevation in donors was associated with early heart-transplant failure (9) and IL-8 elevation in bronchoalveolar lavage fluid with early lung-transplant failure (8). More recently, plasma IL-6 before organ procurement has been associated with lower recipient 6-month hospital-free survival in recipient transplanted with organ (all organs pooled) obtained from 30 brain-dead donors (45), suggesting that cytokine removal by hemoadsorption could be of benefit (46). However, hospital-free survival does not tell us whether or not the cause of death was due to graft failure, and the follow-up is rather short considering the usual follow-up used in transplantation program (e.g., years). Multiorgan failure associated with overwhelming inflammation is characterized by functional, rather than structural, abnormalities and may constitute an adaptive protective mechanism (47). In fact, organ failures in the setting of massive inflammation such as severe sepsis are usually reversible in survivors (47). Inflammation may be associated more closely with early organ failure as opposed to long-term organ failure. In keeping with this hypothesis, renal replacement therapy may tide patients over a phase of early renal transplant dysfunction, as reported in recipients of kidneys from non-heart-beating donors who died after out-of-hospital cardiac arrest (48). Obviously, a larger study is required to figure out if organ dysfunction related to inflammation injury really translates in meaningful difference in outcome later on because most of it seems reversible.
Limitations of the study
We used skeletal muscle abnormalities as a surrogate of alterations occurring in organs retrieved for transplantation. Although data from animal studies support the validity of this approach, we cannot exclude differences between muscle and other organs regarding responses to brain death.
In conclusion, brain death was consistently followed by a systemic inflammatory response with high levels of cytokines and presence of plasma endotoxin regardless of the cause of death. Our brain-dead patients showed evidence of leukocyte reprogramming, a feature previously reported in severe sepsis. RNA expression in muscle biopsies suggested activation of apoptosis. However, the severity of inflammation, as assessed by circulating cytokine levels, was not associated with a lower long-term transplant survival. These data suggest that the inflammation occurring after brain death is reversible after transplantation, and that the massive inflammation found in some patients does not alter graft survival in the recipient. It is conceivable that some of the insults induced by systemic inflammation may in fact induce heat shock protein responses and improve or alter the outcomes from secondary insults.
The authors thank Charlène Blanchet for help in developing the Figure available online as Supplemental Digital Content 2.
1. Wijdicks EF: The diagnosis of brain death. N Engl J Med 344:1215-1221, 2001.
2. Gasser M, Waaga AM, Laskowski IA, Tilney NL: The influence of donor brain death on short and long-term outcome of solid organ allografts. Ann Transplant 5:61-67, 2000.
3. Gridelli B, Remuzzi G: Strategies for making more organs available for transplantation. N Engl J Med 343:404-410, 2000.
4. Wood KE, Becker BN, McCartney JG, D'Alessandro AM, Coursin DB: Care of the potential organ donor. N Engl J Med 351:2730-2739, 2004.
5. Weiss S, Kotsch K, Francuski M, Reutzel-Selke A, Mantouvalou L, Klemz R, Kuecuek O, Jonas S, Wesslau C, Ulrich F, et al: Brain death activates donor organs and is associated with a worse I/R injury after liver transplantation. Am J Transplant 7:1584-1593, 2007.
6. Amado JA, Lopez-Espadas F, Vazquez-Barquero A, Salas E, Riancho JA, Lopez-Cordovilla JJ, Garcia-Unzueta MT: Blood levels of cytokines in brain-dead patients: relationship with circulating hormones and acute-phase reactants. Metabolism 44:812-816, 1995.
7. Fisher AJ, Donnelly SC, Hirani N, Burdick MD, Strieter RM, Dark JH, Corris PA: Enhanced pulmonary inflammation in organ donors following fatal non-traumatic brain injury. Lancet 353:1412-1413, 1999.
8. Fisher AJ, Donnelly SC, Hirani N, Haslett C, Strieter RM, Dark JH, Corris PA: Elevated levels of interleukin-8 in donor lungs is associated with early graft failure after lung transplantation. Am J Respir Crit Care Med 163:259-265, 2001.
9. Wagner FD, Jonitz B, Potapov EV, Qedra N, Wegscheider K, Abraham K, Ivanitskaia EA, Loebe M, Hetzer R: Procalcitonin, a donor-specific predictor of early graft failure-related mortality after heart transplantation. Circulation 104:I192-I196, 2001.
10. Le Gall JR, Lemeshow S, Saulnier F: A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA 270:2957-2963, 1993.
11. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonca A, Bruining H, Reinhart CK, Suter PM, Thijs LG: The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med 22:707-710, 1996.
12. Adrie C, Adib-Conquy M, Laurent I, Monchi M, Vinsonneau C, Fitting C, Fraisse F, Dinh-Xuan A, Carli P, Spaulding C, et al: Successful cardiopulmonary resuscitation after cardiac arrest as a "sepsis like" syndrome. Circulation 106:562-568, 2002.
13. Keselman HJ, Cribbie R, Holland B: Controlling the rate of type I error over a large set of statistical tests. Br J Math Stat Psychol 55:27-39, 2002.
14. Borge BA, Kalland KH, Olsen S, Bletsa A, Berggreen E, Wiig H: Cytokines are produced locally by myocytes in rat skeletal muscle during endotoxemia. Am J Physiol Heart Circ Physiol 296:H735-H744, 2009.
15. Adrie C, Haouache H, Saleh M, Memain N, Laurent I, Thuong M, Darques L, Guerrini P, Monchi M: An underrecognized source of organ donors: patients with brain death after successfully resuscitated cardiac arrest. Intensive Care Med 34:132-137, 2008.
16. Ali AA, Lim E, Thanikachalam M, Sudarshan C, White P, Parameshwar J, Dhital K, Large SR: Cardiac arrest in the organ donor does not negatively influence recipient survival after heart transplantation. Eur J Cardiothorac Surg 31:929-933, 2007.
17. Wilson DJ, Fisher A, Das K, Goerlitz F, Holland BK, De La Torre AN, Merchant A, Seguel J, Samanta AK, Koneru B: Donors with cardiac arrest: improved organ recovery but no preconditioning benefit in liver allografts. Transplantation 75:1683-1687, 2003.
18. Cavaillon JM, Adib-Conquy M, Fitting C, Adrie C, Payen D: Cytokine cascade in sepsis. Scand J Infect Dis 35:535-544, 2003.
19. Kusaka M, Fukami N, Sasaki H, Higuchi T, Ishikawa K, Shiroki R, Hoshinaga K: IP-10, apoptotic genes, and calcineurin subtype messenger RNA kinetics occurring in rat renal isografts from brain-dead donors. Transplant Proc 37:361-363, 2005.
20. Olszyna DP, Prins JM, Dekkers PE, De Jonge E, Speelman P, Van Deventer SJ, Van Der Poll T: Sequential measurements of chemokines in urosepsis and experimental endotoxemia. J Clin Immunol 19:399-405, 1999.
21. Gasser M, Waaga AM, Laskowski IA, Tilney NL: Organ transplantation from brain dead donors: its impact on short and long term outcome revisited. Transplant Rev 15:1-10, 2001.
22. Chen YW, Shi R, Geraci N, Shrestha S, Gordish-Dressman H, Pachman LM: Duration of chronic inflammation alters gene expression in muscle from untreated girls with juvenile dermatomyositis. BMC Immunol 9:43, 2008.
23. Witasp A, Nordfors L, Schalling M, Nygren J, Ljungqvist O, Thorell A: Increased expression of inflammatory pathway genes in skeletal muscle during surgery. Clin Nutr 28:291-298, 2009.
24. Carmo-Araujo EM, Dal-Pai-Silva M, Dal-Pai V, Cecchini R, Anjos Ferreira AL: Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies. Int J Exp Pathol 88:147-154, 2007.
25. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW, et al: The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 276:33293-33296, 2001.
26. Marie C, Losser MR, Fitting C, Kermarrec N, Payen D, Cavaillon JM: Cytokines and soluble cytokine receptors in pleural effusions from septic and nonseptic patients. Am J Respir Crit Care Med 156:1515-1522, 1997.
27. Zhang Y, Pilon G, Marette A, Baracos VE: Cytokines and endotoxin induce cytokine receptors in skeletal muscle. Am J Physiol Endocrinol Metab 279:E196-E205, 2000.
28. Cavaillon JM, Adrie C, Fitting C, Adib-Conquy M: Reprogramming of circulatory cells in sepsis and SIRS. J Endotoxin Res 11:311-320, 2005.
29. Cavaillon JM, Adib-Conquy M, Cloez-Tayarani I, Fitting C: Immunodepression in sepsis and SIRS assessed by ex vivo cytokine production is not a generalized phenomenon: a review. J Endotoxin Res 7:85-93, 2001.
30. Muret J, Marie C, Fitting C, Payen D, Cavaillon JM: Ex vivo T-lymphocyte derived cytokine production in SIRS patients is influenced by experimental procedures. Shock 13:169-174, 2000.
31. Stein AB, Bolli R, Guo Y, Wang OL, Tan W, Wu WJ, Zhu X, Zhu Y, Xuan YT: The late phase of ischemic preconditioning induces a prosurvival genetic program that results in marked attenuation of apoptosis. J Mol Cell Cardiol 42:1075-1085, 2007.
32. Jee SH, Shen SC, Chiu HC, Tsai WL, Kuo ML: Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency. Oncogene 20:198-208, 2001.
33. Kobayashi S, Werneburg NW, Bronk SF, Kaufmann SH, Gores GJ: Interleukin-6 contributes to Mcl-1 up-regulation and TRAIL resistance via an Akt-signaling pathway in cholangiocarcinoma cells. Gastroenterology 128:2054-2065, 2005.
34. Curcio C, Asheld JJ, Chabla JM, Ayubcha D, Hallas BH, Horowitz JM, Torres G: Expression profile of Bag 1 in the postmortem brain. J Chem Neuroanat 32:191-195, 2006.
35. Gotz R, Wiese S, Takayama S, Camarero GC, Rossoll W, Schweizer U, Troppmair J, Jablonka S, Holtmann B, Reed JC, et al: Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells. Nat Neurosci 8:1169-1178, 2005.
36. Homma S, Iwasaki M, Shelton GD, Engvall E, Reed JC, Takayama S: BAG3 deficiency results in fulminant myopathy and early lethality. Am J Pathol 169:761-773, 2006.
37. Mica L, Harter L, Trentz O, Keel M: Endotoxin reduces CD95-induced neutrophil apoptosis by cIAP-2-mediated caspase-3 degradation. J Am Coll Surg 199:595-602, 2004.
38. O'Neill AJ, Doyle BT, Molloy E, Watson C, Phelan D, Greenan MC, Fitzpatrick JM, Watson RW: Gene expression profile of inflammatory neutrophils: alterations in the inhibitors of apoptosis proteins during spontaneous and delayed apoptosis. Shock 21:512-518, 2004.
39. Van de Craen M, Van Loo G, Pype S, Van Criekinge W, Van den brande I, Molemans F, Fiers W, Declercq W, Vandenabeele P: Identification of a new caspase homologue: caspase-14. Cell Death Differ 5:838-846, 1998.
40. Seidelin JB, Nielsen OH: Expression profiling of apoptosis-related genes in enterocytes isolated from patients with ulcerative colitis. APMIS 114:508-517, 2006.
41. Krajewska M, Rosenthal RE, Mikolajczyk J, Stennicke HR, Wiesenthal T, Mai J, Naito M, Salvesen GS, Reed JC, Fiskum G, et al: Early processing of Bid and caspase-6, -8, -10, -14 in the canine brain during cardiac arrest and resuscitation. Exp Neurol 189:261-279, 2004.
42. Birks EJ, Yacoub MH, Burton PS, Owen V, Pomerance A, O'Halloran A, Banner NR, Khaghani A, Latif N: Activation of apoptotic and inflammatory pathways in dysfunctional donor hearts. Transplantation 70:1498-1506, 2000.
43. Kirsch M, Bertrand S, Lecerf L, Ginat M, Garnier JP, Loisance D: Brain death-induced myocardial dysfunction: a role for apoptosis? Transplant Proc 31:1713-1714, 1999.
44. Van Der Hoeven JA, Moshage H, Schuurs T, Nijboer M, Van Schilfgaarde R, Ploeg RJ: Brain death induces apoptosis in donor liver of the rat. Transplantation 76:1150-1154, 2003.
45. Murugan R, Venkataraman R, Wahed AS, Elder M, Hergenroeder G, Carter M, Madden NJ, Powner D, Kellum JA: Increased plasma interleukin-6 in donors is associated with lower recipient hospital-free survival after cadaveric organ transplantation. Crit Care Med 36:1810-1816, 2008.
46. Kellum JA, Venkataraman R, Powner D, Elder M, Hergenroeder G, Carter M: Feasibility study of cytokine removal by hemoadsorption in brain-dead humans. Crit Care Med 36:268-272, 2008.
47. Singer M, De Santis V, Vitale D, Jeffcoate W: Multiorgan failure is an adaptive, endocrine-mediated, metabolic response to overwhelming systemic inflammation. Lancet 364:545-548, 2004.
48. Sanchez-Fructuoso AI, Marques M, Prats D, Conesa J, Calvo N, Perez-Contin MJ, Blazquez J, Fernandez C, Corral E, Del Rio F, et al: Victims of cardiac arrest occurring outside the hospital: a source of transplantable kidneys. Ann Intern Med 145:157-164, 2006.
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Brain death; inflammation; endotoxin; transplantation; graft; apoptosis; heart arrest
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