American Journal of Emergency Medicine (2012) 30, 405-411

Original Contribution

Relation of signal in mononuclear cell with endotoxin response and clinical outcome after trauma^?

Hsin-Chin Shih MD, PhD?, Mu-Shun Huang MD, Chen-Hsen Lee MD

Institute of Emergency and Critical Care Medicine, National Yang-Ming University, Taipei, Taiwan

Division of Surgery, Department of Emergency, Taipei Veterans General Hospital, Taipei, 11217 Taiwan ROC

Received 26 November 2010; accepted 15 December 2010

Abstract

Background: We investigated the correlation of proinflammatory transcript nuclear factor ?B (NF-?B) and antioxidative gene transcript nuclear factor-erythroid 2-related factor 2 (Nrf2) expressions in peripheral blood mononuclear cells (PBMCs) with the tumor necrosis factor ? (TNF-?) response after endotoxin stimulation and the clinical outcome of severely injured patients.

Methods: Thirty-two severe blunt trauma patients (injury severity score N16) with systemic inflammatory response syndrome were enrolled. Age- and sex-matched healthy persons were the controls. Patients’ blood samples were obtained at 24 and 72 hours after injury. Peripheral blood mononuclear cells were isolated, and measurements for NF-?B p65 translocation, Nrf2 and phosphorylated inhibitory ?B-? expressions, and TNF-? levels were assayed after endotoxin stimulation.

Results: In the trauma patients, TNF-? hyporesponse, depressed NF-?B p65 translocation, and phosphorylated inhibitory ?B-? expression in PBMCs were found at 24 and 72 hours after injury; the Nrf2 expressions in PBMCs were not significantly different between patients and controls. The TNF-? levels had significant correlation with the NF-?B translocation and the trend of negative correlation with Nrf2 expression. Fifteen patients had critical injury (injury severity score >=25). Patients with critical injury had a lower NF-?B signal and a lower TNF-? response than did the counter group. Twelve patients developed organ failure; their Nrf2 expressions were significantly lower than those of patients without organ failure. Conclusions: The endotoxin hyporesponse associated with NF-?B and Nrf2 signal alternations in PBMCs of injured patients develops early after injury. The hyporesponse of PBMCs with a lower TNF-? level correlates with a lower NF-?B signal and is associated with critical injury, whereas a depressed Nrf2 expression in PBMCs is associated with later organ failure in trauma patients.

(C) 2012

Introduction

Several studies have shown that trauma induces a cytokine hyporesponse to extracorporeal stimulation, which is as-

^? Financial support was from Taipei Veterans General Hospital and National Science Committee.

* Corresponding author. Division of Surgery, Department of Emer-

gency, Taipei Veterans General Hospital, Taipei City, Taiwan 11217, ROC.

E-mail address: [email protected] (H.-C. Shih).

sumed as immune system anergy and is related to injury severity and the outcome of injured patients [1-3]. Majetschak et al [1] have shown that the extent of traumatic damage determines a graded depression of the endotoxin response of peripheral blood mononuclear cells (PBMCs) from patients with Blunt injuries. Ploder et al [2] reveal that lipopolysaccha- ride (LPS)-induced tumor necrosis factor ? (TNF-?) produc- tion is correlated with survival in septic trauma patients. However, the mechanisms of cytokine hyporesponse will require more investigations. One of the major signal factors

0735-6757/$ - see front matter (C) 2012 doi:10.1016/j.ajem.2010.12.025

responsible for cytokine expressions is the nuclear factor ?B (NF-?B), a redox-sensitive transcript. Cytokine release requires nuclear translocation of NF-?B. Stimuli, such as LPSs, mediate the degradation of inhibitory ?B (I?B) and thus enable the translocation of NF-?B to the nucleus of immune cells [4,5]. In recent reports, nuclear factor-erythroid 2-related factor 2 (Nrf2), another redox-sensitive transcript, is respon- sible for the antioxidant gene expression after stress and may counter the action of NF-?B translocation [6]. Nuclear factor- erythroid 2-related factor 2, a basic leucine zipper redox- sensitive transcription factor, is a pleiotropic protein that regulates the basal and inducible expression of a battery of antioxidant and other cytoprotective genes by binding to a cis-acting enhancer sequence known as the antioxidant re- sponse element (ARE) [7,8]. Under normal conditions, nuclear levels of Nrf2 are low; however, under stress, such as oxidative stimuli, nuclear accumulation of Nrf2 increases, resulting in enhanced transcriptional activation of its targets, which in turn confers protection against various environmental stresses [6-8]. Modulations of transcriptional activities have been proposed as one of the new potential therapeutic targets to improve posttraumatic morbidity and mortality [9]. In our present study, we tried to investigate the correlation of the NF-?B signal and Nrf2 expression in PBMCs with the in vitro TNF-? response after endotoxin stimulation and the clinical outcome of severely injured patients.

Materials and methods

Patients

Adult patients with severe blunt trauma (injury severity score [ISS] N16) and systemic inflammatory response syndrome were subsequently enrolled. Systemic inflamma- tory response syndrome was defined as the presence of at least 2 of the following criteria: temperature lower than 36?C or temperature higher than 38?C, heart rate more than 90 beats/min, respiratory rate more than 20 breaths per minute, PaCO2 less than 32 mm Hg, white blood cell count more than 12 000 cells/mm³ or less than 4000 cells/mm³, or more than 10% immature band forms. Patients with major underlying medical disease or isolated traumatic brain on arrival as well as patients who died of their injuries within the first 72 hours were excluded from the study. All patients were evaluated in the emergency department within 24 hours after injury and reevaluated at 72 hours later. Organ function was assessed daily using the modified score of Lefering et al [10]. Organ function was graded from 0 to 2, where a score of 2 indicates organ failure. Organ failure was determined by a daily evaluation of the involved organ dysfunctions and was graded daily on a scale of 0 to 2. We defined organ failure as at least 1 organ failure (lung, heart, kidney, liver), with a sum of these dysfunctions of 2 or more. Age- and sex-matched healthy volunteers served as controls. This study was approved by the institutional research review board of Taipei

Veterans General Hospital. Written informed consent was obtained from all patients. In case of unconsciousness, a legal representative was asked for consent.

Isolation of PBMCs

Blood samples obtained from patients and controls were diluted 1:1 with phosphate-buffered saline, then layered over Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ, USA) and centrifuged at 400g for 30 minutes. Mononuclear cell layer at the plasma-Ficoll interface was obtained and suspended with cell culture medium (RPMI 1640) containing 10% serum. Cells were enumerated and adjusted to a density of 10⁶ cell/mL. After isolation, cells were used immediately for preparation of nuclear and cytoplasmic extracts or were cultured first for 1 hour at 37?C in a 5% CO₂ incubator in RPMI 1640 medium in the presence of LPS (1 ug per 10⁶ cells/mL, 0111:B4 Lipopolysaccharide; Sigma, St. Louis, MO, USA).

Cytosolic fraction

Freshly collected or cultured PBMCs were washed once with phosphate-buffered saline before extraction. Adherent cells cultured with LPS were harvested with a cell scraper and added to corresponding nonadherent cells. Briefly, cells were homogenized in ice-cold cytosolic lysis buffer (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA,

1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail, which contains, per milliliter, the following ingredients: 500 ug of antipain; 500 ug, aprotinin; 500 ug, leupeptin; 50 ug, pepstatin;

750 ug, bestatin; 400 ug, phosphoramidone; and 500 ug, trypsin inhibitor) using a homogenizer (French press, Constant System, Northants, UK), followed by a 15-minute incubation on ice. It was then centrifuged at 12 000g for

5 minutes at 4?C. From that, the supernatant cytosolic fraction was carefully extracted and stored at -80?C.

Nuclear protein preparation

The nuclear pellet was then resuspended in ice-cold lysis buffer (20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl,

1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail) and was rocked vigorously on a shaking platform for 20 minutes. The nuclear protein fraction was isolated via centrifugation for 10 minutes at 4?C, and the supernatant was harvested and kept at 80?C. Protein concentrations were determined according to the method of Bradford.

Nuclear translocation of NF-B

Nuclear translocation and DNA binding activity of NF-?B were assayed by using enzyme-linked immunosorbent assay

Western blot analysis for phosphorylated”>Total patients		No organ failure	Organ failure	P ?
Patient no.	32	20	12
Sex (female)	8	5	3	.283
Age (y)	33.2 +- 14.0	30.1 +- 13.8	38.6 +- 13.4	.480
ISS	23.2 +- 4.4	22.3 +- 3.6	24.7 +- 5.2	.737
SBP	99.7 +-18.0	105.4 +- 19.9	90.2 +- 8.8	.018
WBC	15 384 +- 3698	15 218 +- 4019	14 942 +- 4666	.412
BD	-3.6 +- 3.8	-2.0 +- 3.3	-6.5 +- 3.0	.018
Transfusion (U)	3.6 +- 1.7	3.2 +- 1.6	4.2 +- 1.8	.152
Mortality	3	0	3	.044
SBP indicates systolic blood pressure; WBC, leukocyte count; BD, Base deficit at emergency department. * P value of comparison between patients with organ failure vs patients without organ failure (no organ failure). Data are presented as mean +- SD.

(ELISA) method. TransFactor kits (BD Mercury Transfactor Assay, BD Biosciences, Palo Alto, CA, USA) identifying DNA-protein interactions were used for rapid detection of NF-?B p65 activities in nuclear extracts according to the manufacturer’s instructions [11,12]. After the addition of chromogen, the plate was read at optic density 650 nm (ELISA reader).

Table 1 Demographics and clinical characteristics of 32 severely injured patients dichotomized by organ failure

Western blot analysis for phosphorylated inhibitory ?B-? and Nrf2

Cytosolic fractions were loaded on 10% polyacrylamide gels in sodium dodecyl sulfate-polyacrylamide gel electro- phoresis sample buffer. Gels were electrophoresed at 150 V for 75 minutes then transferred for 60 minutes onto nitrocellulose membranes. Membranes were blocked for 2 hours at Room temperature, with 5% fat-free Powdered milk in TBST (tris- buffered saline Tween-20). Membranes were incubated in polyclonal rabbit anti-human antibody to phosphorylated inhibitory ?B-? (p-I?B-?) (Cell Signaling Biotechnology, Beverly, MA, USA) or to Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4?C, washed with TBST, and then exposed to a goat antirabbit horseradish peroxidase- conjugated antibody for 1 hour at room temperature. Detection was accomplished by chemiluminescence on Hyperfilm-MP and scan by Laser Scanning Densitometer (Amersham Biosciences, Piscataway, NJ, USA). The scanned images were imported into an image analyzer (ImageQuant, GE Healthcare Bioscience, Uppsala, Sweden). Phosphorylated I?B-? and Nrf2 expressions of patients were determined and normalized with controls.

ELISA for in vitro TNF-?

The TNF levels in the supernatant of cultured PBMCs after LPS stimulation were assayed using a commercially available human TNF-? ELISA kit (R&D Systems, Minnneapolis, MN, USA) in accordance with the manufac- turer’s instructions.

Statistical analysis

All continuous variances were described as mean +- SD in the present study. Statistical analyses were performed using a commercially available statistical software package (SPSS, version 16.0; SPSS, Chicago, IL). For data that were not normally distributed, the Mann-Whitney U test was used. The Spearman rank nonparametric correlation was used to estimate the correlation between 2 values. In case of catego- rical data, Fisher exact test was used. Statistical significance was set at P b .05.

Results

Thirty-two patients were enrolled in the present study;

8 were female. Twelve patients developed organ failure (organ failure score, >=2; average, 3.1 +- 0.7); all of them had respiratory failure, and 3 of them died of multiple organ failure on days 9, 11, and 16 postinjury, respectively. The first blood culture samples were negative for bacterial growth in all patients. The demographics and clinical characteristics of the patients are shown in Table 1. Compared with patients without organ failure, patients with organ failure had

Table 2 Demographics and clinical characteristics of 32 severely injured patients dichotomized by ISS

Total patients ISS >=25 ISS b25 P ?

Patient no. 32 15 17

Sex (female) 8 4 4 .283

Age (y) 33.2 +- 14.0 31.0 +- 10.9 35.2 +- 16.4 .847

SBP 99.7 +- 18.0 99.8 +- 19.6 99.5 +- 15.7 .998

WBC 15 384 +- 3698 15 982 +- 1118 14 352 +- 814 .412

BD -3.6 +- 3.8 -4.0 +- 4.1 -3.3 +- 3.7 .133

Transfusion 3.6 +- 1.7 4.0 +- 2.1 3.2 +- 1.3 .151

(U)

Organ failure 12 9 3 .027

Mortality 3 2 1 .548

* P value of comparison between patients with ISS of 25 or higher vs patients with ISS less than 25. Data are presented as mean +- SD.

24 h			P	72 h P
Controls (n = 32)	118	+- 15.0		128 +- 19.0 b.01
Patients (n = 32)	53.9	+- 23.9		45.9 +- 13.3
ISS >=25 (n = 15)	41.7	+- 15.8	b.01	40.6 +- 14.0 b.01
ISS b25 (n = 17)	64.6	+- 25.1		51.2 +- 10.2
Organ failure (n = 12)	44.5	+- 13.6	.088	44.6 +- 11.8 .279
No failure (n = 20)	59.5	+- 27.2		46.8 +- 14.4
P value: controls vs patients; patients with ISS of 25 or higher vs ISS less than 25; patients with organ failure vs without organ failure (no failure). Data are presented as mean +- SD.

significantly lower emergency department blood pressure, a higher base deficit and higher mortality. Fifteen patients had critical injury (ISS >=25). The demographics and clinical characteristics of patients dichotomized by ISS are shown in Table 2. There was no significant difference with respect to age, blood pressure, and base deficit in the emergency department except significant association with higher rates of organ failure in patients with critical injury.

Table 3 In vitro TNF-? levels (pg/mL) of PBMCs after endotoxin stimulation at 24 and 72 hours after injury

The levels of in vitro TNF-? release, either at 24 or 72 hours postinjury, were significantly suppressed in trauma patients compared with those levels in the controls (Table 3).

A

When subgroup analyses were performed, patients with critical injury (ISS >=25) had lower TNF levels compared with those patients with less severe injury (ISS b25) at 24 and 72 hours after injury. Although patients with organ injury tended to have a lower response at 24 hours after injury, their levels did not reach statistical significance (Table 3).

In the study of the NF-?B signal of PBMC, NF-?B p65 translocation and phosphorylated I-?B were significantly lower in injured patients when compared with those in the controls. The NF-?B signals were found to be significantly lower in the patients with critical injury at 24 and 72 hours after injury when compared with those in the counter groups (Fig. 1). Moreover, there was significant correlation of NF-?B translocation with the in vitro TNF-? levels at 24 and 72 hours after injury (Spearman ? correlation analysis: R = 0.424, P = .016 at 24 hours; R = 0.379, P = .032 at 72 hours; Fig. 3). In patients with or without organ failure, no significant discrep- ancy of NF-?B signal was found between them (Fig. 1).

In the study of Nrf2 expressions, compared with the controls, trauma patients did not have a significant change of Nrf2 expressions. However, when subgroup analysis was based on the development of later organ failure, the expres- sions of Nrf2 were significantly lower in patients with organ failure compared with those in patients without organ failure

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24 hours 72 hours 24 hours 72 hours

Fig. 1 Nuclear factor ?B translocation and p-I?B-? expression at 24 and 72 hours after injury (ratio to control). A, Patients with critical injury had significantly decreased NF-?B signals compared with the counter group (*P b .01; ISS >=25 vs ISS b25). B, There was no significant discrepancy between patients with organ failure (org. failure) or without org. failure (no failure) at 24 and 72 hours after injury.

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LPS produced significantly lower TNF-? levels than did the healthy controls, indicating posttraumatic endotoxin hypor- esponse or tolerance [2,3,13]. A discrepancy was found with respect to the outcomes of endotoxin tolerance between the well-controlled second-hit study of animal and clinical investigations. The animal study showed beneficial effect, whereas the clinical investigation had the opposite finding [14]. The studies of Adib-Conquy et al [15], Heagy et al [3], and Ploder et al [2] showed that patients with decreased TNF-? release by LPS-stimulated blood cells were associ- ated with unfavorable clinical outcomes. In our present study, the endotoxin hyporesponse of TNF-? in PBMCs was found in severely injured patients on the first and third postinjury days. Patients with critical injury (ISS >=25) had a significantly lower response of TNF-? than patients without critical injury (ISS N16 and b25). These findings were in line

B ISS>25 ISS<25

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Fig. 2 Nuclear factor-erythroid 2-related factor 2 expression of PBMC at 24 and 72 hours after severe injury (ratio to control). A, patient with organ failure had significantly higher Nrf2 expressions at 24 and 72 hours than patients without organ failure (no failure) (*P b

.05 organ failure vs no failure). B, Patients with or without critical injury did not have a significant difference in the Nrf2 expression.

both at 24 and 72 hours after injury (Fig. 2). When patients were dichotomized by critical injury, no significant differ- ence in the expression levels of the Nrf2 was found (Fig. 2). In the study of the correlation of Nrf2 and in vitro TNF-? level, no statistical significance was found, although a trend of negative correlation was found at 24 hours after injury (R = -0.323, P = .072 at 24 hours; Fig. 3).

Discussion

Severe injury frequently causes a systemic inflammatory response syndrome that makes the patient more susceptible to infection. These Inflammatory processes induce immuno- suppression, as assessed by the reducED capacity of circu- lating leukocytes from patients with severe injury to produce cytokines upon endotoxin activation. Several studies have shown that monocytes from injured patients stimulated with

with the previous report [1]. In our present study, we found that the endotoxin hyporesponse after injury was not asso- ciated with higher morbidity; however, there was a signifi- cant trend (P = .088; Fig. 3) in the 24 hours after injury. Samples obtained earlier or additional patient studies may show a significant result.

Our study further showed that the endotoxin hypore- sponse with depressed TNF-? release was associated with a lower NF-?B signal. Several studies revealed that the NF-?B activities in monocytes from trauma patients were sup- pressed early after injury [15-19]. The down-regulation of the NF-?B system may have a close relationship with the endotoxin hyporesponse after trauma. Our study supported these findings and highlighted the role of the NF-?B system on endotoxin hyporesponse after injury. Although further studies may be needed, the initial activation of PBMC, which happened very early after trauma, might have depleted NF-?B and resulted in the deactivation or desensitization of PBMC in subsequent endotoxin stimulation [18]. This occurrence could be the result of Proinflammatory cytokines or mediator productions during trauma-related events. These molecules could mimic the effects of endotoxins in vivo and in vitro; they induced tolerance similar to the endotoxin hyporesponse seen in trauma patients [19]. Theoretically, the more severe injury might have incurred a massive cytokine or inflammatory response that depleted the reservoir of the NF-?B signals, which resulted in an aggravated hypore- sponse in subsequent in vitro endotoxin stimulation. Our study showed that NF-?B translocation in PBMCs had a positive correlation with the TNF-? response. These results implied that the NF-?B signal is essential for the TNF-? response upon endotoxin stimulation. Although the Nrf2 had a negative trend of correlation with the endotoxin response, the results did not reach statistical significance.

Although the Nrf2 expressions were found to be no dif- ferent in injured patients when compared with the controls, Nrf2 expressions were found to be significantly lower in patients who developed organ failure in the present study. Nuclear factor-erythroid 2-related factor 2 is the transcrip- tion factor responsible for both the constitutive and inducible

Fig. 3 The scatter graphs of the NF-?B translocations (A) and Nrf2 expressions (B) with the in vitro TNF-? levels in PBMCs after endotoxin stimulation at 24 and 72 hours after injury. Nuclear factor ?B translocation shows significant correlation with the TNF-? levels (Spearman ? correlation analysis: R = 0.424, P = .016 at 24 hours; R = 0.379, P = .032 at 72 hours). The trend of negative correlation of Nrf2 expression and TNF-? levels at 24 hours was without significance (R = -0.323, P = .072 at 24 hours; R = -0.266, P = .141 at 72 hours).

expression of the ARE-regulated genes. Antioxidant re- sponse element is a cis-acting transcriptional regulatory element involved in the activation of genes coding for a number of antioxidant proteins and phase II detoxifying enzymes, including nicotinamide adenine dinucleotide phosphate quinone oxidoreductase, Glutathione peroxidase, ferritin, ?-glutamylcysteine synthetase, and heme oxyge- nase-1 [20,21]. Normally, under basal conditions, Nrf2 is bound to Keap1 in the cytoplasm because of an interaction between a single Nrf2 protein and a Keap1 dimer. Keap1 serves as a substrate linker protein for the interaction of the Cul3-based E3-ubiquitin ligase complex with Nrf2, leading to ubiquitination of Nrf2 and proteosomal degradation [22]. Exposure to several stressors and inducing agents leads to dissociation of Nrf2 from Keap1, thereby rescuing Nrf2 from proteasomal degradation and allowing for entry into the nucleus. These include the endogenous activators such as Reactive oxygen species, and others [23]. Nuclear factor-

erythroid 2-related factor 2 null mice have decreased the basal and inducible expression of antioxidant genes, increased oxidative stress, and decreased reducing activity and antioxidant capacity [6], suggesting that the Nrf2/ARE pathway is critical for the regulation of the intracellular Redox status. In the present study, unlike the NF-?B, critical injury did not have significant influence on the level of Nrf2 expression in PBMCs after endotoxin stimulation compared with the same expression level in patients without critical injury. On the other hand, our study showed that patients with organ failure had significantly lower Nrf2 expressions than patients without organ failure. The present study did not explore the mechanism of these changes on Nrf2 expres- sions. For more patients with shock and higher base deficit found in the group with organ failure, these physiologic derangements might incur more oxidative stress. It is possi- ble that this hyperoxidative stress induced early activation of the Nrf2 signal, which became tolerant to subsequent in

vitro endotoxin stimulation with less Nrf2 expression. De- fective Nrf2 expression under such unfavorable conditions might result in inadequate endogenous antioxidant protec- tion and lead to later organ failure in trauma patients. Our study also showed that lower Nrf2 is more important than lower NF-?B of PBMC in the prediction of organ failure after severe injury.

The interactions of the NF-?B and Nrf2 pathways have recently been under investigation. Data have shown that NF-?B p65 may participate in the negative regulation of Nrf2/ARE signaling [24]. Moreover, it has recently been shown that Nrf2 is a critical regulator of NF-?B activation both by modulating I?B degradation and MyD88-dependent and MyD88-independent signaling [25]. In our present study, there seemed to be an opposite effect of the NF-?B p65 signal and Nrf2 expression in PBMC on the TNF-? levels from endotoxin stimulation, whereas there was no significant correlation of these 2 signals in the present study, indicating that their interactions are complicated.

There were several limitations within our present study. We did not measure the translocation of the Nrf2 directly. The increased expression of Nrf2 may not reflect its true translocation and transcription status; however, increased expression of the Nrf2 indicated the lower degradation of this protein and the greater possibility of the translocation. The other concern is the clinical outcome with respect to mortal- ity. Our mortality cases were too few to determine the asso- ciation of signals with mortality. Moreover, we did not investigate the mechanisms of these signal alternations; it is unclear exactly how the early suppressed Nrf2 expression results in later organ failure in trauma patients. Finally, the Nrf2 expression in PBMC might not reflect the status in individual organs such as in the lungs. Despite these con- cerns, our results showed that measures to enhance the Nrf2 signal may be helpful for patients with severe injury.

In conclusion, the endotoxin hyporesponse associated with NF-?B and Nrf2 signal alternations in PBMC of a patient develops early after injury. Hyporesponse of PBMC with a lower TNF-? level correlates with a lower NF-?B signal and is associated with critical injury, whereas depressed Nrf2 expression in PBMC is associated with later organ failure in trauma patients. These signal alternations may serve as the predictors of clinical outcome and targets for the immunomodulation after severe injury.

References

1. Majetschak M, Flach R, Kreuzfelder E, et al. The extent of traumatic damage determines a graded depression of the endotoxin responsive- ness of peripheral blood mononuclear cells from patients with blunt injuries. Crit Care Med 1999;27(2):313-8.
2. Ploder M, Pelinka L, Schmuckenschlager C, et al. Lipopolysaccharide- induced tumor necrosis factor alpha production and not monocyte human leukocyte antigen-DR expression is correlated with survival in septic trauma patients. Shock 2006;25(2):129-34.
3. Heagy W, Hansen C, Nieman K, et al. Impaired ex vivo lipopoly- saccharide-stimulated whole blood tumor necrosis factor production

may identify “septic” intensive care unit patients. Shock 2000;14(3): 271-6.

1. Shih HC, Wang CY, Wen YS, et al. Spleen artery embolization aggravates endotoxin hyporesponse of peripheral blood mononuclear cells in patients with spleen injury. J Trauma 2010;68(3):532-7.
2. Wang H, Ma S. The cytokine storm and factors determining the sequence and severity of organ dysfunction in Multiple organ dysfunction syndrome. Am J Emerg Med 2008;26(6):711-5.
3. Thimmulappa RK, Lee H, Rangasamy T, et al. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest 2006;116(4):984-95.
4. Marzec JM, Christie JD, Reddy SP, et al. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB 2007;21(9):2237-46.
5. Rangasamy T, Guo J, Mitzner WA, et al. Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med 2005;202:47-59.
6. Coimbra R, Melbostad H, Loomis W, et al. Phosphodiesterase inhibition decreases nuclear factor-kappaB activation and shifts the Cytokine response toward anti-inflammatory activity in acute endotox- emia. J Trauma 2005;59(3):575-82.
7. Lefering R, Goris RJ, Van Nieu, et al. Revision of the multiple organ failure score. Arch Surg 2002;387:14-20.
8. Benotmane AM, Hoylaerts MF, Collen D, et al. Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium. Anal. Biochem 1997;250:181-5.
9. Shen Z, Peedikayil J, Olson GK, et al. Multiple transcription factor profiling by enzyme-linked immunoassay. BioTechniques 2002;32: 1168-74.
10. Munoz C, Carlet J, Fitting C, et al. Dysregulation of in vitro cytokine production by monocytes during sepsis. J Clin Invest 1991;88:1747-54.
11. Shih HC, Wei YH, Lee CH. Magnolol alters the course of endotoxin tolerance and provides early protection against endotoxin challenge following sublethal hemorrhage in rats. Shock 2004;22(4):358-63.
12. Adib-Conquy M, Asehnoune K, Moine P, et al. Long-term-impaired expression of nuclear factor-?B and Ik?alpha in peripheral blood mononuclear cells of trauma patients. J Leukoc Biol 2001;70:30-8.
13. Adib-Conquy M, Adrie C, Moine P, et al. NF-kappaB expression in mononuclear cells of patients with sepsis resembles that observed in lipopolysaccharide tolerance. Am J Respir Crit Care Med 2000;162(5): 1877-83.
14. Blackwell TS, Blackwell TR, Christman JW. Induction of endotoxin tolerance depletes nuclear factor-kappaB and suppresses its activation in rat alveolar macrophages. J Leuko Biol 1997;62:885-91.
15. Biberthaler P, Stegmaier J, Mayer V. Initial posttraumatic translocation of NF-kappaB and TNF-alpha mRNA expression in peripheral blood monocytes of trauma patients with multiple injuries: a pilot study. Shock 2004;22:527-32.
16. Cavaillon JM. The nonspecific nature of endotoxin tolerance. Trends Microbiol 1995;3:320-4.
17. Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med 2004;10:549-57.
18. Tong K, Katoh Y, Kusunoki H, et al. Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model. Mol Cell Biol 2006;26:2887-90.
19. Cullinan S, Gordan J, Jin J, et al. The keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase. Mol Cell Biol 2004;24:8477-86.
20. Dinkova-Kostova AT, Holtzclaw WT, Kensler TW. The role of keap1 in cellular protective responses. Chem Res Toxicol 2005;18:1779-91.
21. Ma Q, Battelli L, Hubbs AF. Multiorgan autoimmune inflammation, enhanced lymphoproliferation, and impaired homeostasis of reactive oxygen species in mice lacking the antioxidant-activated transcription factor Nrf2. Am J Pathol 2006;168:1960-74.
22. Liu GH, Qu J, Shen X. NF-kappaB/p65 antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitating recruitment of HDAC3 to MafK. Biochem Biophys Acta 2008;1783:713-27.