Transcription Factor NFκB Expression and Postsurgical Organ Dysfunction

Abstract

Objective

To examine the role of neutrophil NFκB activation in organ dysfunction after major surgery.

Summary Background Data

NFκB is a transcription factor involved in the signal transduction of many stimuli that may participate in the pathogenesis of sepsis and resultant multiple organ dysfunction syndrome (MODS). It may therefore be a potential target for modulation in the reduction of postsurgical MODS.

Methods

Twenty-five patients undergoing major vascular surgery (thoracoabdominal aortic aneurysm repair) were studied. Perioperative levels of neutrophil NFκB, CD11b, and glutathione were measured. In vitro inhibition experiments using NFκB inhibitors were also performed.

Results

No differences in clinical parameters were apparent before surgery between the patients who subsequently developed MODS and those who did not. However, there was a significant difference in preoperative levels of NFκB between the patients who developed postoperative organ dysfunction and those who did not. There was also a significant preoperative difference between patients who survived surgery and those who did not. Glutathione levels were reduced both in patients who developed MODS and those who did not at the onset of surgery. NFκB inhibitors suppressed patient plasma-stimulated NFκB activation in healthy neutrophils.

Conclusions

Preoperative neutrophil NFκB status may be a marker of postoperative outcome after major surgery, and therapy aimed at attenuating neutrophil NFκB activation may reduce postoperative sepsis and organ dysfunction.

Multiple organ dysfunction syndrome (MODS) is a term used to describe the sequential failure of single organ systems leading ultimately to a general failure of multiple organs. ¹ MODS is responsible for 50% to 80% of all deaths in the surgical intensive care unit and has remained so for the past 20 years. ^2–4 It develops secondary to severe systemic inflammation (sepsis), in which the activated neutrophil plays a central role. ^4–6 The neutrophil is therefore subject to intensive investigation as a target for modulation in the reduction of MODS. ^7–9

Various strategies have been used to reduce neutrophil activation in both experimental and clinical MODS. Because neutrophil activation usually involves ligation of surface receptors, ¹⁰ most strategies have tried to target various receptors or ligands. Such strategies have met with limited success clinically, ^11–14 which probably reflects the complex nature of neutrophil activation in clinical MODS, where the neutrophil is stimulated by a variety of simultaneous extracellular signals, including various cytokines and endotoxin.

A common event occurring as a result of many ligand–receptor interactions is the activation of the transcription factor NFκB (Fig. 1). NFκB is a dimer of two DNA binding subunits (RelA and p50). Inactive NFκB exists in the cytoplasm bound to IκB, which prevents NFκB from translocating to the nucleus and binding DNA. NFκB activation is the process by which NFκB is dissociated from ΙκB and translocated to the nucleus, where it binds DNA and ultimately initiates transcription. This very complex process occurs rapidly (within minutes of extracellular stimulation) and involves phosphorylation, proteolysis, and redox changes.

Figure 1. Hypothetical pathway for NFκB activation. Any one of a number of extracellular inducers results in the activation of a number of protein kinases that phosphorylate IκB/p105 (shown in red; RelA, dark blue; p50, light blue). Phosphorylated IκB/p105 is recognized by ubiquitin conjugating enzymes. Ubiquitinated IκB/p105 is degraded and free NFκB then translocates to the nucleus. Activation of NFκB can be interrupted at different stages. Antioxidant treatment blocks phosphorylation of IκB/p105 and proteasome inhibitors block degradation of IκB/p105.

NFκB is rapidly activated in response to many pathologic signals that may be relevant during surgical trauma, including cytokines, ^15,16 adhesion molecules, ^17,18 endotoxin, ¹⁹ hypoxia, ^20,21 and shear stress. ²² These stimuli activate several different protein kinases (protein kinase C, MAP kinase, ceramide kinase, IκB kinase) ²³ that initiate NFκB activation. It has been postulated that the common mechanism by which these signals result in the activation of NFκB is by a change in the redox status of the cells. ^24,25 Increased levels of hydrogen peroxide have been implicated in IκB degradation and NFκB DNA binding. The increase in intracellular reactive oxygen species (hydrogen peroxide) is also accompanied by a depletion of antioxidants such as glutathione. ²⁶ It has been reported that replenishing glutathione levels by treatment with the antioxidant N-acetyl-L-cysteine (NAC) reduces activation of NFκB. ²⁷ Other antioxidants, including pyrrolidine dithiocarbamate (PDTC) and diethyldithiocarbamate (DDTC), have also been shown to abrogate activation of NFκB. ²⁸

Activation of NFκB results in the transcription of genes that can participate in the inflammatory reaction by inducing the production of cytokines, immunoreceptors, and cell adhesion molecules. ²⁹ This transcription factor is therefore a potentially attractive target for immunomodulation.

Information on the role of NFκB in human sepsis and MODS is limited but shows that the activity of this transcription factor is increased during inflammation and sepsis. ^30,31 There is, however, no published information about neutrophil NFκB status and the development of organ dysfunction. In this study we investigated the role of neutrophil NFκB activation in the development of postsurgical MODS and potential modulation of neutrophil NFκB activation in the prevention of MODS-related neutrophil activation.

METHODS

Sample Collection

Measurement of nuclear levels of NFκB has recently been facilitated by the development of a new assay ³² that measures the level of expression of intranuclear NFκB and therefore “activated” NFκB. Venous blood samples were collected from 25 healthy volunteers (19 women, 6 men, age range 19–54 years, median 32) to assess baseline values of NFκB in this normal population and also to use in in vitro studies of NFκB activation and suppression. The samples were collected into EDTA tubes (Becton Dickinson, Abington, UK) and were immediately processed for analysis of neutrophil nuclear NFκB expression.

The development of postsurgical organ dysfunction is a serious risk after surgery for the repair of thoracoabdominal aortic aneurysm (TAAA), ^33,34 so this type of surgery serves as a good clinical model for the study of MODS. The development of organ dysfunction after this kind of surgery has been shown to be associated with increased intraoperative neutrophil activation, which may occur in response to cytokines, endotoxin, and free radicals. Twenty-five patients (7 women, 18 men, age range 17–80 years, median 65) undergoing elective repair of TAAA were included in the study. Routine preoperative assessments (urea and electrolyte tests, creatinine baseline, renal MAG-3 scan, blood gas and spirometry, stress echocardiogram, full blood count, erythrocyte sedimentation test, fasting lipids and glucose, liver function tests, clotting screen, ankle/brachial pressure indices, thoracoabdominal computed tomography, aortic calibrated angiography, and full history, including smoking status) were performed and recorded for all patients. One patient had Crawford ³⁵ type I aneurysm, 5 had Crawford type II, 4 had Crawford type III, and 15 had Crawford type IV aortic aneurysms. Blood (EDTA, Becton Dickinson) was obtained from patients before surgery, 30 minutes after aortic cross-clamping, and at regular intervals after visceral reperfusion. Samples were collected and processed immediately for flow cytometric analysis for surface CD11b expression, nuclear NFκB expression, and intracellular glutathione levels. Plasma was stored at -70°C for batch analysis.

Postoperative Systemic Inflammatory Status and Clinical Complications

All patients were staged for signs of systemic inflammation and sepsis using the systemic inflammatory response syndrome scoring system. ¹ Median values for temperature, heart rate, respiratory rate, and white blood cell counts were obtained for the first 5 days after surgery, and patients were scored for systemic inflammatory response syndrome as described. For the purpose of this study, postoperative renal dysfunction was defined as an increased plasma creatinine level (>200 μM/L) and the need for postoperative hemodialysis. Respiratory dysfunction was defined as hypoxia with a need for prolonged (>5 days) mechanical ventilatory support. Cardiac dysfunction was defined as cardiac failure or myocardial infarction. Neurologic dysfunction was defined as the presence of transient ischemic attacks, stroke, or paraplegia.

Measurement of Neutrophil NFκB Activation

Neutrophil NFκB expression was measured as described by Foulds. ³² Briefly, a preparation of nuclei was first obtained from anticoagulated lysed whole blood by treatment in detergent for 10 minutes (using reagents contained in the Cycletest PLUS DNA reagent kit, Becton Dickinson). Rabbit polyclonal NFκB antibody (40 μL RelA, Santa Cruz Biological, Heidelberg, Germany) was then added for 10 minutes, followed by a further 10-minute incubation with 2.5 μL fluorescein isothiocyanate (FITC)-conjugated antirabbit monoclonal antibody (Sigma, Poole, UK). Two hundred microliters of cold propidium iodide solution was then added to the nuclei, and the preparation was incubated for 10 minutes. The nuclei were then acquired on a flow cytometer (FACScan, Becton Dickinson) using Lysis II with the doublet discrimination module activated. Analysis was performed using Lysis II analysis software (Becton Dickinson). A singlet gate was first set up and the nuclei from polymorphonuclear cells were then gated. This population was analyzed for FITC staining using histogram analysis. The mean channel fluorescence was used as an indicator of the intensity of nuclei fluorescence.

Measurement of Neutrophil CD11b Expression

A 100-μL aliquot of anticoagulated blood was added to a 10-μL aliquot of phycoerythrin-conjugated anti-CD11b monoclonal antibody (Becton Dickinson) and incubated at room temperature for 15 minutes in the dark. Erythrocytes were then lysed using FACS lysing solution for 20 minutes in the dark. Samples were then washed in Dulbecco’s phosphate-buffered saline (PBS; Sigma). All samples were run through a flow cytometer (FACScan, Becton Dickinson) using Lysis II software within 2 hours of collection. Measurement of neutrophil CD11b expression was carried out using Lysis II analysis software. Briefly, the granulocyte population was gated on by virtue of its light-scattering properties, and this gate was then plotted as a frequency histogram of red fluorescence. Results are expressed as arithmetic mean fluorescence, an indicator of the intensity of fluorescence.

Measurement of Intracellular Glutathione

Intracellular levels of neutrophil glutathione concentration were measured by flow cytometry using a method adapted from that described by Treumer and Valet. ³⁶ One hundred microliters of anticoagulated (EDTA) whole blood was deposited in a Falcon 2025 tube (Becton Dickinson). Two milliliters of FACS lysing solution was added to the blood and the tube was then incubated at room temperature for 20 minutes. The cells were then washed two times in PBS (1,500 rpm for 5 minutes) and resuspended in 500 μL PBS and 5 μL O-phthaldialaldehyde (stock made up at 100 mmol/L in DMSO). The cells were then incubated in the dark for 5 minutes and then acquired on the flow cytometer using Lysis II acquisition software. Analysis was carried out using Lysis II analysis software. A neutrophil gate was first demarcated using the FSC/SSC settings. This neutrophil gate was then viewed as a red frequency histogram using the FL2 setting. The arithmetic mean channel fluorescence was used as an indicator of the intensity of fluorescence.

In Vitro NFκB Inhibition Studies

Cocultures of neutrophils from healthy donors and plasma from the TAAA group were set up as follows. Five-milliliter blood samples were obtained from donors in EDTA tubes and washed five times in PBS. Washed blood was made up to 5 mL with PBS, and 100 μL of this was aliquoted into a Falcon 2500 tube. One hundred-microliter samples (taken at the following time points: preoperative, clamp on, 30 minutes after clamp on, clamp off, 10, 30, 50, and 60 minutes after start of reperfusion) of plasma obtained from a TAAA patient was added to the cells and the tube was incubated at 37°C for 30 minutes. Cells were then washed three times with PBS, were resuspended in 100 μL PBS, and were then processed for NFκB determination as described above. Inhibition experiments were performed by preincubating blood preparations for 1 hour with PDTC, DDTC, or NAC (all Sigma) at varying concentrations before stimulation with TAAA plasma, NFκB staining, and flow cytometric analysis. Inhibition experiments using the NFκB SN50 cell-permeable inhibitor peptide (Calbiochem, Nottingham, UK) were performed as follows. One hundred microliters of washed lysed healthy whole blood was incubated with various concentrations of the SN50 peptide or a control peptide (SN50M cell-permeable inactive control peptide, Calbiochem) for 15 minutes at room temperature and then challenged with 100 μL endotoxin (10 ng/mL) or plasma from TAAA patients for 30 minutes at room temperature. Cells were then processed for NFκB determination.

Statistical Analysis

Statistical analysis was performed with Statview 4.0 (Abascus Concepts, Inc., CA) and GraphPad Instat Mac (GraphPad software, San Diego, CA). Mann–Whitney tests were used to assess the statistical differences between groups (uncomplicated vs. complicated). Repeated-measures analyses of variance were used to assess differences between several perioperative measurements in NFκB. Post-hoc analysis to evaluate differences in NFκB expression between groups at single time points was done by modified t tests (Bonferroni). Linear regression was used to assess whether preoperative NFκB expression was predictive of subsequent intraoperative neutrophil activation.

RESULTS

NFκB Levels in Healthy Volunteers

Neutrophil nuclear NFκB levels in the 25 healthy volunteers studied were found to be similar, with a mean channel fluorescence of 75.08 (SEM = 1.6). There were no age- or sex-related trends in this small population.

NFκB Status and Postsurgical MODS

Of the 25 TAAA patients studied, 6 died within 4 hours of surgery, 9 recovered from surgery without additional postoperative support other than routine ventilation for the first 24 hours after surgery, and 10 developed organ dysfunction (6 MODS, 4 single organ dysfunction). Six patients who developed organ dysfunction died within 30 days of surgery. There were no apparent preoperative clinical differences between the patients with postsurgical organ dysfunction and those without. The patients with postsurgical organ dysfunction (complicated group) had increased intraoperative neutrophil activation, demonstrated by high levels of the adhesion receptor CD11b, as previously reported. ^33,34

There were notable differences in NFκB status between patients with postsurgical complications and those without (Fig. 2). The most important revelation from the data were the differences in the preoperative levels of neutrophil NFκB between the patients with complications and those without (P = .0015, Mann–Whitney test). The mean in the uncomplicated group was 97.55 (SEM = 12.8); the mean in the complicated group was 210.1 (SEM = 27). There was also a difference in preoperative NFκB levels between patients with organ failure who survived surgery and those who did not (Fig. 3, P = .0190, Mann–Whitney test). The mean in the survivors was 140.5 (SEM = 29); the mean in the nonsurvivors was 256.5 (SEM = 28). There was significant correlation between preoperative nuclear NFκB expression and peak intraoperative CD11b surface expression (R = 0.775, P = .0001) (Fig. 4).

Figure 2. Changes (means, SEM) in neutrophil nuclear NFκB expression between patients who developed complications (•) and those who did not (○) . Time points shown are before surgery (P-OP), clamp on and 30 minutes into clamp time (C-0, C-30), start of reperfusion and 10, 30, 50, and 60 minutes after the start of reperfusion (R-0, R-10, R-30, R-50, R-60). P = .0015, Mann-Whitney, difference preoperative uncomplicated vs. complicated.

Figure 3. Changes (means, SEM) in neutrophil nuclear NFκB expression (mean channel fluorescence [MCF]) in patients who developed complications and survived (□) and those who did not (▪). Time points shown are before surgery (P-OP), clamp on and 30 minutes into clamp time (C-0, C-30), start of reperfusion and 10, 30, 50, and 60 minutes after the start of reperfusion (R-0, R-10, R-30, R-50, R-60). P = .012, analysis of variance.

Figure 4. Regression line between preoperative levels of NFκB and CD11b expression (mean channel fluorescence [MCF]) at 50 minutes after the start of reperfusion. (○), patients who recovered without complications. (□), patients who developed complications but survived. (▪), patients who died of complications. R = 0.775, P = .0001.

Both groups of patients showed a similar increase in NFκB activation immediately after the start of surgery (P < .05, Bonferroni). It was hypothesized that this increase was due to a change in the redox status of the neutrophil. Because such changes are reflected by depletion of antioxidants such as glutathione, ²⁶ intracellular glutathione levels were measured. There were no differences between perioperative levels of neutrophil intracellular glutathione between the patients with complications and those without (Fig. 5). There was, however, a significant decrease in the level of intracellular glutathione soon after the start of surgery in both groups of patients; this was apparent at the first intraoperative sampling (P < .01, Bonferroni).

Figure 5. Changes (means, SEM) in neutrophil intracellular glutathione levels (mean channel fluorescence [MCF]) between patients who developed complications (•) and those who did not (○). Time points shown are before surgery (P-OP), clamp on, and at 0, 30, and 50 minutes of reperfusion (R-0, 30, 50).

Modulation of NFκB to Reduce Neutrophil Activation

Given that the level of NFκB activation was different between patients who developed organ dysfunction after surgery versus those who did not, modulating neutrophil NFκB levels may help reduce neutrophil activation and potentially reduce neutrophil-related organ dysfunction. The putative NFκB inhibitors PDTC, DDTC, NAC, and SN50 cell-permeable inhibitor peptide ³⁷ were tested for their ability to reduce neutrophil NFκB activation in healthy neutrophils stimulated by plasma from TAAA patients.

Inhibition of neutrophil NFκB expression was observed on preincubation of healthy neutrophils with all four anti-NFκB agents before challenge with peak intraoperative CD11b activating plasmas from the TAAA patients. DDTC, PDTC, and NAC inhibited NFκB at previously published doses. ^27,28 NAC neutralized NFκB upregulation at 30 mmol/L (Fig. 6;P = .001, paired t test), and DDTC and PDTC abrogated expression of NFκB at 1mmol/L (Fig. 7,P < .01, Student paired t test).

Figure 6. Effect of the antioxidant N-acetyl-L-cysteine (NAC) on NFκB stimulation in neutrophils from healthy volunteers. First bar (C) shows mean (SEM) baseline NFκB expression (mean channel fluorescence [MCF]) in nuclei from neutrophils isolated from healthy volunteers. Subsequent bars shows NFκB expression in these neutrophils when stimulated with LPS for 30 minutes lipopolysaccharide; stimulated with peak plasmas from patients who subsequently developed complications (plasma); and pretreated with 1, 10, and 30 mmol/L NAC before stimulation with the peak plasma. P < .05 (Student paired t test) at 10 and 30 mmol/L NAC.

Figure 7. Effect of diethyldithiocarbamate (DDTC) (upper graph) and pyrrolidine dithiocarbamate (PDTC) (lower graph) on NFκB stimulation in neutrophils from healthy volunteers. First bar (C) shows mean (SEM) baseline NFκB expression in nuclei from neutrophils isolated from healthy volunteers. Subsequent bars show NFκB expression in these neutrophils stimulated with LPS for 30 minutes lipopolysaccharide; stimulated with peak plasma from patients who subsequently developed organ dysfunction (Plasma); and pretreated with 0.1 and 1 mmol/L drug before stimulation with the peak plasma. P < .05 (Student paired t test) at 1 mmol/L drug.

Experiments with the NFκB SN50 cell-permeable inhibitor peptide (Fig. 8), which inhibits subcellular traffic of NFκB from the cytoplasm to the nucleus, showed that this peptide was also capable of inhibiting the activation of NFκB in healthy neutrophils in response to activation by TAAA plasma at a concentration of 50 and 100 μg/mL peptide. The control peptide, which can also traverse the cell membrane but has no inhibitory action, had no effect on NFκB activation.

Figure 8. Effect of permeable inhibitor peptide SN50 and inactive control peptide (light bars) on NFκB stimulation in neutrophils from healthy volunteers. First bar (C) shows mean (SEM) baseline NFκB expression in nuclei from neutrophils isolated from healthy volunteers. Subsequent bars show NFκB expression in these neutrophils: stimulated with LPS for 30 minutes lipopolysaccharide; stimulated with peak plasma from patients who developed complications (Plasma); and pretreated with 10, 30, 50, and 100 mg/mL SN50 (dark bars) or control peptide (light bars) before stimulation with the peak plasma.

DISCUSSION

Controlling the overwhelming inflammatory response that results in sepsis and organ failure remains an important issue in critical care medicine. Therapies that target individual mechanisms, no matter how relevant, are unlikely to succeed, as proven when such therapies have been tested in clinical trials. Strategies aimed at targeting more general pathways that affect several mechanisms are more likely to reduce the inflammatory response resulting in sepsis and MODS.

NFκB is a ubiquitous transcription factor involved in the signal transduction pathway of many inducers of the inflammatory response ²⁹ and is therefore a potentially attractive target for immunomodulation to reduce sepsis and organ dysfunction. This study has shown that levels of nuclear-bound NFκB (activated NFκB) ³² are greater in patients who develop organ dysfunction after TAAA surgery. Patients with lower levels of nuclear NFκB recovered from surgery without organ dysfunction.

TAAA surgery is a complicated procedure that involves temporary cross-clamping of the aorta while repair is being performed, rendering the visceral organs temporarily ischemic. This can have profound biologic effects, with both local and systemic effects resulting in cell death and tissue injury. ^38,39 This study shows that TAAA surgery results in significant oxidative stress in both patients who develop organ dysfunction and those who do not; the patients who developed complications did not have higher levels of oxidative stress than patients who recovered without complications. This was reflected in the intraoperative NFκB status in the patients, which showed slight increases in both groups. Replenishing glutathione levels with NAC prevented NFκB activation in healthy cells stimulated with plasma from TAAA patients, suggesting that intraoperative increases in NFκB activation may be redox-sensitive. Further evidence attesting to redox involvement in NFκB activation during TAAA repair was provided by the in vitro experiments showing the ability of the antioxidants PDTC and DDTC to suppress the NFκB activating ability of plasma from TAAA patients.

Perhaps the most important finding of this study was the marked differences in preoperative neutrophil NFκB levels between the patients who subsequently had organ dysfunction and those who did not. The TAAA patients who had relatively low expression of neutrophil NFκB before surgery had more favorable outcomes than those with higher preoperative levels of NFκB, who tended to develop postoperative complications. Patients with high levels of neutrophil nuclear NFκB were also less likely to recover from TAAA reconstructive surgery.

There is only one published investigation of NFκB activation in sepsis. ³¹ That study investigated activity of NFκB in nuclear extracts from peripheral blood mononuclear cells of 15 patients with sepsis, of whom 10 survived. NFκB activity was measured on days 1, 2, 3, 4, 5, 6, 8, 10, and 14 after admission where available. The day 1 measurement was defined as 100% and other values were calculated as the percentage of day 1. All patients with an NFκB binding activity exceeding 200% of day 1 died. This small study concluded that NFκB activation may be an important event in clinical sepsis.

The present study shows a correlation between preoperative levels of NFκB and the levels of expression of the adhesion receptor CD11b at 50 minutes after the start of reperfusion. Previous studies have shown that the neutrophil CD11b receptor is an intraoperative marker of outcome after both major vascular and nonvascular surgery. ^33,34,40 Also, the preoperative level of neutrophil CD11b has been identified as a marker of clinical outcome in two sets of patients with TAAAs. ³⁴ These two sets comprised patients who died during surgery and patients with extensive aneurysms who subsequently developed organ dysfunction. It was suggested that there may be a population of TAAA patients with “active” neutrophils who react adversely to surgical insult. However, preoperative expression of CD11b could not be used as a general marker for outcome after TAAA surgery, because this marker failed to identify clinical outcome in patients with less extensive aneurysms. This suggests that there may be a set of patients with “primed” rather than “active” neutrophils who react to subsequent stimuli with exaggerated neutrophil activation, characterized by increased levels of neutrophil CD11b receptor. This study shows that preoperative neutrophil NFκB expression is a preoperative marker of organ dysfunction after TAAA surgery. It is postulated that this transcription factor identifies a set of patients with primed neutrophils who mount an exaggerated response (demonstrated by increases in CD11b expression) to subsequent inflammatory signals.

Primed cells may be thought of as being “ready to go” but awaiting further stimulus before the oxidative burst is elicited. ⁴¹ Evidence of primed neutrophils has been presented in the literature. ^42–47 In a series of experiments, McCall et al ^48,49 observed that neutrophils isolated from patients with acute bacterial infections were “toxic” compared with neutrophils from healthy volunteers. These toxic neutrophils exhibited increased oxidative metabolism, phagocytosis, and chemotaxis. Primed neutrophils generate enhanced levels of reactive oxidants and have higher levels of degranulation and greater phagocytic activity compared with untreated cells.

The metabolic response (oxidative burst) to agonistic challenge of neutrophils has been noted to be heterogeneous in a population of normal humans. ⁵⁰ This heterogeneity may be more marked in the population of surgical patients, in which a subset of “anergic” patients has been identified. ^51,52 These studies have suggested that surgical anergic patients are more likely to die of sepsis as a result of intravascular priming of neutrophils. There may be several explanations for such variability in neutrophil responsiveness, including membrane changes occurring because of diet, lifestyle, age, sex, genetic background, surgical disease, and differences in receptor expression on the neutrophils. For example, studies on neutrophil CD16 receptor expression have shown that the constitutive level of surface CD16 expression varies widely within and between persons ^53,54 and that CD16 expression is significantly higher before surgery in patients who develop postoperative sepsis. ⁴⁶

In conclusion, our results show that in our clinical model of MODS (TAAA repair), extensive surgery is associated with a redox-sensitive activation of the transcription factor NFκB. However, the most significant finding of this study was the difference in the preoperative levels of neutrophil NFκB between patients who recovered from surgery without complications and those who developed organ dysfunction after surgery. This makes neutrophil NFκB a potentially important preoperative marker of postoperative organ dysfunction. Further studies aimed at modulating preoperative NFκB status are warranted to assess the role of NFκB activation in the pathophysiology of organ dysfunction and also to assess the role of NFκB modulation in the prevention of neutrophil activation and organ dysfunction after surgery.