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Published ahead of print on June 28, 2007, doi:10.1164/rccm.200609-1250OC
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American Journal of Respiratory and Critical Care Medicine Vol 176. pp. 602-609, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200609-1250OC

Original Article

Gene Expression Profiling Identifies C/EBP{delta} as a Candidate Regulator of Endotoxin-induced Disseminated Intravascular Coagulation

Sjoukje H. Slofstra1, Angelique P. Groot1, Maartje H. P. Obdeijn1, Pieter H. Reitsma1, Hugo ten Cate2 and C. Arnold Spek1

1 Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and 2 Department of Internal Medicine, Academic Hospital and Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands

Correspondence and requests for reprints should be addressed to Sjoukje H. Slofstra, Ph.D., Center for Experimental and Molecular Medicine, G2-132, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: s.h.slofstra{at}amc.uva.nl


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: A runaway inflammatory response to systemic infection or severe trauma is characterized by the activation of a diversity of pathways, ultimately resulting in the development of disseminated intravascular coagulation (DIC) and multiorgan failure.

Objectives: Despite increased fundamental knowledge of the pathogenesis of DIC, the exact molecular mechanisms remain elusive. We aimed therefore to improve our understanding of the molecular pathways underlying endotoxin-induced DIC.

Methods: We performed large-scale gene expression profiling in the liver of mice during the onset of endotoxin-induced DIC. The relevance of an identified candidate gene involved in endotoxin-induced DIC was subsequently assessed in the generalized Shwartzman reaction.

Measurements and Main Results: Approximately 5% of over 20,000 genes were differentially regulated. In addition to well-established sepsis-associated genes, such as macrophage inflammatory protein 1, plasminogen activator inhibitor 1, CD14, and A20, we identified several novel candidates for inflammatory disease of which the transcription factor C/EBP{delta} (CAAT/enhancer binding protein {delta}) was studied further. Induction of DIC in C/EBP{delta}-deficient mice decreased endotoxin-induced systemic inflammation as compared with wild-type mice, as evident from decreased plasma levels of tumor necrosis factor-{alpha} and IL-6. In addition, C/EBP{delta} deficiency partly protected against DIC-induced mortality. Interestingly, C/EBP{delta} deficiency seemed mainly protective by improving renal function. This latter notion was confirmed in an experimental model of renal ischemia/reperfusion injury in which C/EBP{delta} deficiency reduced ischemia/reperfusion-induced creatinine and urea levels.

Conclusions: Our results endorse the usefulness of gene expression profiling in identifying novel mediators of DIC by showing that C/EBP{delta} regulates specific pathologic features of this endotoxin-induced syndrome.

Key Words: microarray • endotoxemia • ischemia/reperfusion • transcription factor



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Despite increased fundamental knowledge of the pathogenesis of disseminated intravascular coagulation (DIC), the exact molecular mechanisms of inflammatory DIC remain elusive.

What This Study Adds to the Field
We identified the transcription factor C/EBP{delta} as a regulator of endotoxin-induced DIC, as well as DIC-induced mortality and renal dysfunction.

 
Sepsis is a clinical syndrome characterized by the systemic production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha} and interleukins IL-6, IL-1beta, and IL-8. The excessive production of these proinflammatory cytokines is sometimes more dangerous than the original stimulus. This is especially notable in severe sepsis, in which the excessive production of proinflammatory cytokines causes capillary leakage, tissue injury, and disseminated intravascular coagulation (DIC). DIC is a syndrome characterized by systemic activation of blood coagulation leading to the formation of intravascular thrombi and impaired organ perfusion. Simultaneously, massive and ongoing activation of coagulation may result in depletion of platelets and coagulation factors, which precipitates bleeding (1). However, despite increased fundamental knowledge of the pathogenesis of DIC, the exact molecular mechanisms remain elusive and consequently therapeutic interventions are rather unsuccessful.

Most clinical trials that aim to counteract the pathologic hallmarks of DIC in patients presenting with severe sepsis target extracellular mediators of the coagulation cascade, such as antithrombin (2) and recombinant tissue factor pathway inhibitor (3), or the inflammatory response (e.g., with inhibitors of TNF-{alpha}) (4, 5). Unfortunately, most of these studies failed to show efficacy and did not provide evidence for a survival benefit. A positive exception appears to be a phase III trial of recombinant human activated protein C (rhAPC). This anticoagulant might also induce direct cellular effects influencing inflammatory gene expression (6–8) and has been proven beneficial in a group of highly selected patients (9). However, even in this highly selected patient panel, more than 24% of patients still died of the consequences of sepsis, indicating that rhAPC is not the final answer in counteracting sepsis-induced organ damage. Additional insight into the molecular mechanisms responsible for organ damage during sepsis-induced DIC is thus warranted.

The liver appears to be one of the key organs that is susceptible to, and responsible for, the initiation of organ failure during DIC/sepsis (10). It is the main organ for bacterial scavenging, inactivation of bacterial products, the clearance and production of inflammatory mediators (11), and the synthesis and release of coagulant factors. Hepatic dysfunction is therefore not only an important hallmark for DIC but also promotes and/or worsens multiple organ dysfunction during sepsis (12, 13).

The generalized Shwartzman reaction is a well-established experimental animal model of endotoxin-induced DIC, which is elicited by two consecutive endotoxin injections (14). The first endotoxin injection results in the priming of certain aspects of the inflammatory response, thereby rendering the host more susceptible to a secondary endotoxin challenge (15, 16), subsequently resulting in (histo-)pathologic lesions resembling human DIC (e.g., accumulation of microthrombi, platelet aggregation, vascular occlusion, inhibition of fibrinolysis, neutrophil accumulation, endothelial injury, and variable degrees of [ischemia-induced] apoptosis and necrosis in the microvasculature; reviewed in Reference 17).

Herein we studied endotoxin-induced differential gene expression in the liver of mice during the onset of DIC. We aimed at the identification and validation of new mediators contributing to the pathogenesis of DIC. Using the Shwartzman reaction, we identified the transcription factor C/EBP{delta} (CAAT/enhancer binding protein {delta}) as a candidate regulator of endotoxin-induced DIC. Interestingly, C/EBP{delta} deficiency seems mainly protective in that it improves renal function.

Some of these results have been previously reported in the form of an abstract (18).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
For the gene expression experiments, 10-week-old, wild-type C57Bl/6 mice were purchased from Harlan Sprague-Dawley, Inc. (Horst, The Netherlands). C/EBP{delta}-deficient (C/EBP{delta}–/–) mice, which were backcrossed more than 10 times to C57Bl/6 mice, were a kind gift from Dr. Esta Sterneck from the National Cancer Institute (Frederick, MD). Age- and sex-matched C57Bl/6 control mice were purchased from Charles River Laboratories (Wilmington, MA). Animal procedures were performed in compliance with the Institutional Standards for Humane Care and Use of Laboratory Animals. The Animal Care and Use Committee of the Academic Medical Centre (Amsterdam, The Netherlands) approved all experiments.

Experimental Design
Generalized Shwartzman reaction as an experimental model for DIC.
The generalized Shwartzman reaction is a well-known and widely accepted model for DIC (14, 15, 19) and is elicited by two consecutive injections of Serratia marcescens endotoxin (Sigma-Aldrich, St. Louis, MO). A priming injection, 5 µg endotoxin in 40 µl sterile saline, was given intradermally in the foot, which was followed 24 hours later by an intravenous challenge injection of 300 µg endotoxin in 100 µl sterile saline (7). Sterile saline–treated mice were killed as control animals and labeled t = –24 hours. For gene expression profiling, wild-type mice (n = 3 per time point) were killed 24 hours after the priming injection of endotoxin (t = 0) or 2 hours after the induction of the Shwartzman reaction by the second endotoxin injection (t = 2). After anesthesia by intraperitoneal injection of 0.007 ml/g FFM mixture (fentanyl, 0.315 mg/ml; fluanisone, 10 mg/ml [Janssen, Beersen, Belgium]; midazolam, 5 mg/ml [Roche, Mijdrecht, The Netherlands]), blood was drawn by heart puncture with a sterile syringe, and transferred to tubes containing heparin (Becton-Dickinson, Alphen aan de Rijn, The Netherlands). Finally, lungs, kidneys and livers were removed and stored in liquid nitrogen.

C/EBP{delta} deficiency and DIC.
C/EBP{delta}-deficient mice and C57BL/6 control animals (n = 8 per group, experiments were repeated three times) were subjected to the generalized Shwartzman reaction as described above and killed 6 hours after the second endotoxin injection. For survival experiments, C/EBP{delta}-deficient (n = 20) and representative wild-type control animals (n = 21) were subjected to a lethal Shwartzman reaction (as described above) and monitored for 30 hours.

Renal ischemia/reperfusion injury model.
Renal ischemia/reperfusion was induced as described previously (20). Briefly, renal arteries were clamped for 45 minutes using microaneurysm clamps through a midline abdominal incision under general anesthesia. After clamp removal, kidneys were inspected for restoration of blood flow. The abdomen was closed in two layers, and all mice received a subcutaneous injection of 50 µg/kg buprenorphin (Temgesic; Schering-Plough, Amstelveen, The Netherlands) for analgesic purposes and were allowed to recover from surgery for 12 hours at 28°C in a ventilated stove. To maintain fluid balance and volume status, mice were supplemented with 1 ml sterile saline intraperitoneally. Sham-operated mice underwent the same procedure without clamping. All mice (n = 6 per group) were killed 1 day after surgery, blood was collected, and kidneys were harvested for further analysis.

RNA Preparation
Total RNA was isolated from snap-frozen tissue using guanidine isothiocyanate/chloroform extraction (guanidine isothiocyanate, Trizol; Gibco, Gaithersburg, MD) followed by precipitation with 2-propanol. After washing with 75% ethanol, the isolated RNA was dissolved in Rnase-free water. Subsequently, high-quality RNA for gene expression profiling was prepared using the RNeasy system (Qiagen, Venlo, The Netherlands).

RNA Fluorescent Labeling and Hybridization
The quality of isolated mRNA was checked using an Agilent 2100 bioanalyzer with an RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA). Subsequently, 20 µg of high-quality RNA was used for fluorescent cyanine (Cy)-3 or Cy5 labeling according to the Agilent Fluorescent Direct Label kit protocol. A pool of RNA of three livers at t = 0 hours was used as a reference sample and was labeled with Cy5. RNA of liver samples at t = –24 hours, 0 hours, and 2 hours was labeled with Cy3 (n = 3 per time point). Labeled RNA samples were hybridized to Agilent Mouse Oligo Microarray slides according to the manufacturer's protocol. After washing, the microarray slides were analyzed at 10-µm resolution using an Agilent scanner and software equipped with automatic spot finding and flagging ability in addition to reporting of Cy3 and Cy5 signal and background for each spot. Raw ratio gene expression data were primarily analyzed using Rosetta Resolver Gene Expression Data Analysis System (Rosetta Biosoftware, Seattle, WA). Time-point biological replicates (n = 3) were combined to create mean intensity ratios and one-way error-weighted analysis of variance with a threshold of P < 0.01 resulted in 3,787 sequences that were differentially regulated.

Evaluation of C/EBP{delta} mRNA Levels by Real-Time Polymerase Chain Reaction
Total RNA was reverse transcribed using random hexamers (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Paisley, UK) according to the supplier's recommendations. Murine C/EBP{delta} mRNA levels were quantified using real-time polymerase chain reaction (PCR) on a LightCycler apparatus (Roche, Mannheim, Germany) using the FastStart DNA Master SYBR Green I kit and 0.8 µl of cDNA. The following primers were used: 5'-ATCGCTGCAGCTTCCTATGT-3' and 5'-AGTCATGCTTTCCCGTGTTC-3'. Expression levels were normalized to the expression level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA. Real-time PCR was performed for 40 cycles consisting of 15 seconds at 95°C, 5 seconds at 57°C, and 20 seconds at 72°C.

C/EBP{delta} Protein Expression Levels
Tissue homogenates were made using a tissue homogenizer in 9 volumes of ice-cold cell lysis buffer (75 mM NaCl, 7.5 mM Tris, 0.5 mM MgCl2, 0.5 mM CaCl2, 0.5% Triton, AEBSF [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride], ethylenediaminetetraacetic acid, peptastatin and leupeptin). One volume of tissue lysate was mixed with 1 volume sodium dodecyl sulfate sample buffer and brought onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. After electrophoresis, protein was transferred onto Immobilon-PVDF membranes (Millipore, Billerica, MA). Membranes were blocked in 1% bovine serum albumin (Sigma-Aldrich) in TBS/0.1% Tween-20 (TBST) for 1 hour. Rabbit polyclonal C/EBP{delta} antibody (sc-306; Santa Cruz Biotechnology, Santa Cruz, CA) was diluted to 1:200 in TBST and membranes were incubated overnight. Rabbit polyclonal beta-actin (sc-1616 Santa Cruz) was diluted to 1:500 in 0.5% bovine serum albumin in TBST. After 1 hour incubation in 1:1,000 anti-rabbit horseradish peroxidase–conjugated secondary antibody (DakoCytomation, Glostrup, Denmark), blots were imaged using LumiLight Plus ECL (Roche, Basel, Switzerland) on a Lumi-imager (Fuji LAS-3000; Fuji, Düsseldorf, Germany) and relative intensities (normalized for beta-actin expression) were determined.

Cytokine Determination in Plasma
IL-6 and TNF-{alpha} were measured using the BD Cytometric Bead Array Mouse Inflammation Kit (Becton-Dickinson) following the manufacturer's instructions. Detection limits were 10 pg/ml.

Kidney and Liver Damage Assay
Creatinine, blood urea nitrogen (BUN) and transaminases in plasma were determined with commercially available kits (Sigma-Aldrich) using a Hitachi analyzer (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.

Statistical Analysis
Statistical analyses were conducted using GraphPad Prism version 3.00 (GraphPad Software, San Diego, CA). Data from successive animal experiments were pooled and levels in control animals were set at 100%. Data are expressed as means ± SEM. Comparison between groups was performed using two-tailed Mann-Whitney tests. Survival curves were compared by a two-tailed log-rank test.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene Expression Profiling
Hepatic gene expression in response to endotoxin was determined at t = –24 hours (baseline), t = 0 hours (24-h post–endotoxin priming), and t = 2 hours (2-h post–endotoxin challenge) in comparison to a reference sample (pool of hepatic mRNA at t = 0 h). Time-point biological replicates (n = 3) were combined to create mean intensity ratios, and one-way error weighted analysis of variance with a threshold of P < 0.01, resulted in 3,787 sequences that were significantly regulated across the indicated time points (see Table E1 of the online supplement).

Differential response to endotoxin infusions.
To identify genes of which the expression was decidedly regulated by endotoxin (either the priming and/or challenge infusion), we compared relative gene expression ratios 24 hours after endotoxin priming (t = 0 h) with baseline levels (t = –24 h) and gene expression levels 2 hours after the endotoxin challenge (t = 2 h) with t = 0 hours. mRNA sequences with a greater than twofold change across time points were selected for further analysis (1,097 sequences [see Table E2]; 2,690 genes were less than twofold regulated across different time points).

Trend patterns.
To discriminate genes regulated by either the priming or challenging endotoxin infusion, differentially regulated genes were grouped into eight trend patterns depending on a descending, ascending, or no response to each administration of endotoxin. As shown in Figure 1, the majority of genes that were regulated in the experimental DIC model were affected by the primary endotoxin injection (678 genes were either increased or decreased in response to endotoxin priming, whereas 419 were not regulated by the priming reaction), which reflects the complexity of the cellular hepatic response to even relatively small amounts of endotoxin. To induce the Shwartzman reaction and thus the DIC phenotype, both endotoxin injections were required. However, relatively few genes were differentially regulated by both endotoxin infusions. As shown in Figure 1, only 39 genes were up-regulated by both injections.


Figure 1
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  		Figure 1. Gene regulation in response to endotoxin. Differentially expressed genes with a greater than twofold change across time points assigned to eight trend patterns featuring decreased, increased, or no response to priming and/or challenge in the Shwartzman reaction. A total of 2,690 genes were less than twofold regulated across different time points (no response/no response; not included in Figure 1).

 
A close inspection of the genes in the increase/increase trend pattern uncovered that the expression of well-known and important players in sepsis and/or endotoxemia was up-regulated by both endotoxin injections. Table 1 shows a subset of genes from this pattern (ranked according to the fold change in response to the second endotoxin challenge). In addition to well-known markers of sepsis, such as macrophage inflammatory protein (MIP)-1, plasminogen activator inhibitor (PAI)-1, KC (murine IL-8 homolog), CD14, and A20, several potential new markers of sepsis induced DIC were identified: C/EBP{delta}, phospholipid scramblase 1 (Plscr1), B-cell translocation gene 2 (Btg2), Traf2 binding protein (T2bp), and olfactory receptor 56 (Olfr56).


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  		TABLE 1. UP-REGULATED GENE EXPRESSION UPON ENDOTOXIN PRIMING AND CHALLENGE (INCREASE/INCREASE TREND PATTERN)

 
Because C/EBP{delta} was suggested to be an important player in inflammation (21), we further focused on the role of this transcription factor in endotoxin-induced DIC.

C/EBP{delta} and Endotoxin-induced DIC
C/EBP{delta} expression in lung and kidney.
The liver is obviously not the only organ affected by sepsis-induced DIC, and impaired respiratory and renal functions are additional pathologic hallmarks. Therefore, we determined C/EBP{delta} expression levels in both lung and kidney during endotoxin-induced DIC. As shown in Figure 2, C/EBP{delta} mRNA up-regulation in response to endotoxin was not restricted to the liver but was also observed in lung (Figure 2A) and kidney (Figure 2B). C/EBP{delta} mRNA levels were increased more than 10-fold 2 hours after the challenge reaction in both lung and liver. Six hours after the endotoxin challenge, C/EBP{delta} mRNA levels in kidney were even further induced.


Figure 2
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  		Figure 2. C/EBP{delta} expression in lung (A) and kidney (B) as determined by real-time polymerase chain reaction during the Shwartzman reaction (n = 3). C/EBP{delta} expression was determined in relation to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Bars represent mean ± SE.

 
C/EBP{delta} protein expression.
To determine whether C/EBP{delta} protein expression was likewise increased as C/EBP{delta} mRNA expression, protein levels were determined in tissue homogenates of lung, kidney, and liver. Figure 3 demonstrates that the induction of the Shwartzman reaction results in three- to fourfold increased expression of the C/EBP{delta} protein. In lung and kidney tissue, the expression was high up to 6 hours after the induction of the Shwartzman reaction, whereas C/EBP{delta} expression started to decrease at this time point in the liver samples.


Figure 3
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  		Figure 3. C/EPB{delta} protein expression in lung (A), kidney (B), and liver (C) homogenates was determined by Western blotting using a C/EPB{delta}-specific antibody. Protein levels are expressed relative to control beta-actin levels. Baseline levels and levels at 2 and 6 hours after the endotoxin challenge injection in the Shwartzman reaction are shown (n = 3). Bars represent mean ± SEM.

 
C/EBP{delta} deficiency and inflammation.
To determine the role of C/EBP{delta} in inflammation during endotoxin-induced DIC, wild-type and C/EBP{delta}-deficient mice were subjected to the Shwartzman reaction. At baseline (t = –24 h) and 6 hours after the challenge endotoxin infusion (t = –6 h), systemic levels of the proinflammatory cytokines TNF-{alpha} and IL-6 were measured. No significant differences in baseline levels were observed between wild-type and C/EBP{delta}-deficient mice. As shown in Figure 4, the induction of the Shwartzman reaction caused a significant increase in plasma TNF-{alpha} and IL-6 levels. Figure 4A shows that, in challenged mice, C/EBP{delta} deficiency resulted in lower TNF-{alpha} levels (49.3 ± 7.7%) compared with those in wild-type mice (100 ± 13.2%) (p < 0.01). Similarly, IL-6 levels in challenged C/EBP{delta}-deficient mice were 45.6 ± 6.0% of the levels observed in wild-type mice (100 ± 11.2%) (p < 0.0001) (Figure 4B).


Figure 4
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  		Figure 4. C/EBP{delta} deficiency down-regulates the inflammatory response. Systemic tumor necrosis factor (TNF)-{alpha} (A) and IL-6 (B) levels in wild-type (wt) and C/EBP{delta}-deficient mice at baseline (t = –24 h) and 6 hours after the endotoxin challenge (t = 6 h; Shwartzman reaction). Proinflammatory cytokines after the induction of the Shwartzman reaction are representative of three successive experiments (n = 8 per group). Bars represent mean ± SE. **p < 0.01; *** p < 0.001.

 
Effect of C/EBP{delta} on organ damage.
To obtain further insight into the role of C/EBP{delta} in endotoxin-induced DIC, we compared markers of kidney and liver failure between wild-type and C/EBP{delta}-deficient mice. As shown in Figures 5A and 5B, baseline plasma levels of markers that are indicative for kidney injury (i.e., creatinine and BUN) were not significantly different between the two mouse strains. Induction of the Shwartzman reaction increased creatinine levels almost twofold in wild-type mice but did not increase creatinine levels in C/EBP{delta}-deficient mice (100 ± 17.1% for wild-type mice and 56.3 ± 9.5% for C/EBP{delta}-deficient mice at t = 6 h, p = 0.03). As shown in Figure 5B, BUN levels also increased due to the endotoxin infusions, although to a lesser extent in the C/EBP{delta}-deficient mice (C/EBP{delta}–/–, 81.3 ± 6.7%, vs. wild-type, 100 ± 3.9%; p = 0.03). Overall, these results suggest that C/EBP{delta}-deficient mice are protected against kidney damage provoked by endotoxin-induced DIC.


Figure 5
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  		Figure 5. Effect of C/EBP{delta} deficiency on pathologic hallmarks of disseminated intravascular coagulation. Analysis of organ damage markers creatinine (A) and blood urea nitrogen (BUN) (B) representing kidney damage, and ALAT (alanine aminotransferase) (C) and ASAT (aspartate aminotransaminase) (D) representing liver damage in plasma of mice (n = 8 per strain) 6 hours after the induction of the Shwartzman reaction. Values are depicted as mean % ± SEM (representative of three experiments). (E) Survival curve of wild-type (wt) (n = 20) and C/EBP{delta}-deficient (n = 21) mice after the induction of the Shwartzman reaction.

 
In contrast to kidney damage, hepatic injury, which was assessed by measuring plasma levels of the transaminases ASAT (aspartate aminotransferase) and ALAT (alanine aminotransaminase), was not reduced as a result of C/EBP{delta} deficiency. As shown in Figure 5D, ASAT levels were increased in wild-type and C/EBP{delta}-deficient mice during the course of endotoxin-induced DIC (100 ± 8.0% for wild-type and 154.6 ± 22.8% for C/EBP{delta}-deficient mice, p = 0.22). ALAT levels were increased in wild-type and C/EBP{delta}-deficient mice in response to endotoxin (Figure 5C). ALAT levels in C/EBP{delta}-deficient mice were significantly higher compared with wild-type mice (100 ± 11.8% and 181.8 ± 23.9%, p = 0.01). C/EBP{delta} deficiency thus seems not to protect against endotoxin-induced liver injury, and might actually aggravate hepatic damage.

C/EBP{delta} and death in endotoxin-induced DIC.
To establish whether or not the decreased systemic inflammatory profile in combination with improved renal function protects C/EBP{delta}-deficient mice against endotoxin-induced death, we elicited a lethal Shwartzman reaction in wild-type and C/EBP{delta}-deficient mice, and scored mortality over a period of 30 hours. As shown in Figure 5E, all wild-type mice died within 21 hours, whereas 14% of the C/EBP{delta}-deficient mice survived the experiment. The median survival time was delayed by 15% in C/EBP{delta}-deficient mice compared with wild-type mice (p = 0.06). Thus, despite a significant improvement in (acute) inflammation and renal damage, C/EBP{delta}-deficient mice were only moderately protected against endotoxin-induced death. Possibly, augmented liver damage as a result of C/EBP{delta} deficiency limits the protection of C/EBP{delta} deficiency.

Renal Ischemia/Reperfusion-induced Injury and C/EBP{delta}
The opposing effects of C/EBP{delta} deficiency in the systemic Shwartzman model (i.e., protection against kidney damage but aggravation of liver injury) (Figure 5) predict that C/EBP{delta} deficiency should also be protective in a specific model of kidney failure. To test this prediction, we subjected mice to a well-known inflammatory kidney model, renal ischemia/reperfusion. As shown in Figure 6A, the induction of ischemia/reperfusion increased C/EBP{delta} expression levels in wild-type mice, suggesting that C/EBP{delta} may indeed play a role in this model. Subsequent analysis of BUN (Figure 6B) and creatinine (Figure 6C) levels showed that ischemia/reperfusion induced significant kidney damage in wild-type mice. As expected, C/EBP{delta}-deficient mice were protected against ischemia/reperfusion-induced organ damage (creatinine levels of 100 ± 17.6% and 43.2 ± 25.3%, p = 0.03, and urea levels of 100 ± 12.7% and 40.2 ± 15.2%, p = 0.02, for wild-type and C/EBP{delta}-deficient mice, respectively).


Figure 6
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  		Figure 6. C/EBP{delta} expression in renal ischemia/reperfusion (I/R) injury. C/EBP{delta} expression in kidney (A) as determined by real-time polymerase chain reaction after induction of I/R injury (or a sham operation) compared with baseline levels (n = 3 per group). C/EBP{delta} expression was determined in relation to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Kidney damage markers blood urea nitrogen (BUN) (B) and creatinine (C) were determined in wild-type (wt) and C/EBP{delta}-deficient mice 1 day after the induction of I/R injury (n = 6 per group).

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In patients with severe systemic inflammatory disease such as sepsis, DIC is an indicator of poor clinical outcome and probably a determinant of multiple organ failure. Although many elements of the inflammation–procoagulant pathway are now known, novel therapeutic interventions are only partially successful. Clinical trials targeting the coagulation cascade show negative results for antithrombin (2) and recombinant tissue factor pathway inhibitor (3), whereas rhAPC treatment is only considered of benefit in a subset of patients with severe sepsis (9). It is thus fair to conclude that our concepts regarding the important role of coagulation factors in DIC and sepsis are incomplete due to simplification and lack of knowledge.

To boost fundamental knowledge of the pathogenesis of DIC and to identify novel mediators involved in DIC, we subjected mice to the Shwartzman reaction. In this classical animal model of DIC, we determined the kinetics of gene expression in the liver during the onset of endotoxin-induced DIC. The infusion of endotoxin in two consecutive injections (a priming reaction at t = –24 h and an endotoxin challenge at t = 0 h) induced the differential regulation of about 5% (3,787) of the approximately 20,000 genes/transcripts present on the microarray slide. Grouping the differentially regulated genes in trend patterns depending on their response to the consecutive endotoxin administrations showed that the low-dose priming injection already changed the expression pattern of the vast majority of genes (Figure 1). However, DIC is only induced when the priming is followed by a high-dose endotoxin challenge (14). Only 39 genes were actually induced by both the priming and the endotoxin challenge and this group contained several genes known to be associated with sepsis (Table 1). The most prominent of these genes were MIP-1{alpha} and MIP-1beta (22, 23), PAI-1 (24, 25), KC (murine IL-8 homolog) (26), CD14 (27, 28), and A20 (29, 30). In addition to these well-established sepsis-associated genes, the increase-increase trend pattern contained several genes (Plscr1, Btg2, T2bp, Olfr56, and C/EBP{delta}) that were not directly associated with sepsis/DIC.

Plscr1 is a family member of membrane proteins proposed to be involved in the reorganization of plasma membrane phospholipids, but its actual cellular function and role in physiology remain largely unknown (31). The Btg2 gene is known as an immediate growth response gene particularly involved in cell proliferation and differentiation (32, 33), and a direct link to sepsis/DIC is difficult to foresee. The T2bp gene encodes Traf2 binding protein that binds Traf2, which is a key molecule involved in TNF signaling (34). Traf2 binding protein may act as an activator of transcription factors NF-{kappa}B and AP-1 (35), suggesting a possible functional role for Traf2 binding protein in the inflammatory response. Next, Olfr56, better known as IFN-{gamma} inducible protein 47 is an IFN-{gamma}–induced GTP protein with essential pathogen-specific roles in resistance against infection (36). It is thus well conceivable that this gene indeed plays an important role during the development of sepsis/DIC. However, we decided to focus our attention on the final gene in this trend pattern, C/EBP{delta}.

C/EBP{delta} is a member of the C/EBP family of transcription factors and its expression is typically low in most cell types, but it is rapidly induced by a variety of extracellular stimuli, including IL-1, IL-6, endotoxin, and TNF-{alpha} (21). Several in vitro results suggest that C/EBP{delta} is an important player in the inflammatory response. For instance, C/EBP{delta}-binding motifs have been identified in the regulatory regions of various inflammatory genes, including those encoding the cytokines IL-6, IL-8, IL-1beta, and TNF-{alpha} (37, 38), and genes encoding proteins important for macrophagic or granulocytic functions such as inducible nitric oxide synthase, lysozyme, myeloperoxidase. and neutrophil elastase (21). Furthermore, C/EBP{delta} expression levels have been connected to the magnitude of the inflammatory response toward endotoxin (39). In addition, C/EBP{delta} seems a major player in the acute-phase response and C/EBP{delta} regulates cyclooxygenase-2 (COX-2) expression in alveolar epithelial cells (40). Recently, C/EBP{delta} has been identified as a master regulator of proapoptotic gene regulation during the initiation of physiologic cell death (41). Thus, C/EBP{delta} is a transcription factor that is involved in at least two important physiologic processes (inflammation and apoptosis) that are known to play an essential role in acute disorders such as sepsis and DIC. Because intervening at the transcriptional level shuts down several (redundant) inflammatory and apoptotic pathways simultaneously, we hypothesized that C/EBP{delta} targeting might be an attractive strategy to limit the pathologic consequences of endotoxin-induced DIC.

To test this hypothesis, we examined the role of C/EBP{delta} deficiency in endotoxin-induced DIC by subjecting wild-type and C/EBP{delta}-deficient mice to the Shwartzman reaction. C/EBP{delta}-deficient mice display no overt phenotype, are fertile, and achieve normal life spans. The null mutation does, however, lead to altered learning and memory function (42). Our experiments showed that C/EBP{delta} expression was increased upon endotoxin-induced injury in liver as well as kidney and lung (Figure 2). C/EBP{delta} might therefore affect the degree of injury of different organs known to be essential in the pathology of DIC. Subsequently, we showed that mice deficient in C/EBP{delta} are protected against systemic inflammation, as evident from reduced endotoxin-induced cytokine levels (Figure 4) and that C/EBP{delta} deficiency protects to some extent against endotoxin-induced death (Figure 5E). Remarkably, C/EBP{delta}-deficient animals showed diminished endotoxin-induced renal failure (Figures 5A and 5B) but aggravated hepatic injury (Figures 5C and 5D). The increased transaminase levels in plasma of C/EBP{delta}-deficient animals, indicative of augmented liver dysfunction, might be explained by the fact that C/EBP{delta} induces the transcription of most liver-derived class I acute-phase proteins, such as haptoglobin, {alpha}1-acid glycoprotein (43), complement component 3 (44), C-reactive protein (45), and serum amyloid A (46). These acute-phase proteins are known to affect the procoagulant and antifibrinolytic state and may limit proteolytic activity and tissue damage (10), suggesting that C/EBP{delta} deficiency would aggravate hepatic injury.

Despite the prothrombotic features of the generalized Shwartzman model we used, in which there is clear evidence of thrombi in liver and kidney at 6 hours after LPS challenge (14), there was no evidence of systemic coagulation activity at 24 hours after priming or at the time of killing (6 h after the challenging reaction; i.e., t = 30 h) (thrombin-antithrombin complex [TAT] and D-dimer levels below detection level, not shown). Although this may seem unexpected, it is known that the procoagulant effects of LPS in blood, indicated by activation markers, are short lived (47, 48). Furthermore, it is well conceivable that, during the course of the Shwartzman reaction, TAT and D-dimer levels are mainly detectable in the first hours after the priming reaction, but no experimental data to prove or refute this suggestion have been published. Future experiments sampling blood at regular intervals during the time course of the Shwartzman reaction might clarify this issue, but it is nevertheless evident that these blood markers do not further distinguish any differences in mouse responses at the late time points in this study.

The opposite effects of C/EBP{delta} deficiency in the Shwartzman model (i.e., protection against kidney damage but aggravation of liver injury) predict that C/EBP{delta} deficiency would also be protective in a specific model of inflammatory kidney disease. Indeed, C/EBP{delta}-deficient mice were clearly protected against renal injury in a well-established model of renal ischemia/reperfusion injury. Further experiments using different experimental models of inflammatory disease either targeting single or multiple organs should further clarify the organ-specific effect of C/EBP{delta}.

Although surprising at a first glance, discrepant responses between organs (liver and kidney in our study) are a recurring theme in sepsis, and can be attributed to the fact that different cell populations and subtypes have differential sensitivities and altered responses to injury-inducing stimuli (49). In line with this notion, C/EBP{delta} has highly diverse functions that depend on cell type and stimulus (41). Gene expression and functional profiling of wild-type and C/EBP{delta}-deficient hepatic and renal cell lines in response to endotoxin would contribute substantially to a better understanding of the observed differential response.

In conclusion, we aimed at the identification and validation of new mediators contributing to the pathogenesis of DIC. Using gene expression profiling in the Shwartzman reaction, we identified the transcription factor C/EBP{delta} as a candidate regulator of endotoxin-induced DIC. Interestingly, C/EBP{delta} deficiency seems mainly protective in that it improves renal function.


   Acknowledgments
 
The authors thank Joost Daalhuisen, Marieke ten Brink, and Gwen Teske for expert technical assistance.


   FOOTNOTES
 
Supported by a grant from the Netherlands Organization of Scientific Research (to C.A.S.). H.t.C. was a Clinical Established Investigator of the Netherlands Heart Foundation.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200609-1250OC on June 28, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 1, 2006; accepted in final form June 28, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Levi M, ten Cate H. Disseminated intravascular coagulation. N Engl J Med 1999;341:586–592.[Free Full Text]
  2. Warren BL, Eid A, Singer P, Pillay SS, Carl P, Novak I, Chalupa P, Atherstone A, Penzes I, Kubler A, et al. Caring for the critically ill patient: high-dose antithrombin III in severe sepsis: a randomized controlled trial. JAMA 2001;286:1869–1878.[Abstract/Free Full Text]
  3. Abraham E, Reinhart K, Opal S, Demeyer I, Doig C, Rodriguez AL, Beale R, Svoboda P, Laterre PF, Simon S, et al. Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial. JAMA 2003;290:238–247.[Abstract/Free Full Text]
  4. Cohen J, Carlet J. INTERSEPT: an international, multicenter, placebo-controlled trial of monoclonal antibody to human tumor necrosis factor-alpha in patients with sepsis. International Sepsis Trial Study Group. Crit Care Med 1996;24:1431–1440.[CrossRef][Medline]
  5. Opal S, Laterre PF, Abraham E, Francois B, Wittebole X, Lowry S, Dhainaut JF, Warren B, Dugernier T, Lopez A, et al. Recombinant human platelet-activating factor acetylhydrolase for treatment of severe sepsis: results of a phase III, multicenter, randomized, double-blind, placebo-controlled, clinical trial. Crit Care Med 2004;32:332–341.[CrossRef][Medline]
  6. Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001;276:11199–11203.[Abstract/Free Full Text]
  7. Slofstra SH, van't Veer C, Buurman WA, Reitsma PH, ten Cate H, Spek CA. Low molecular weight heparin attenuates multiple organ failure in a murine model of disseminated intravascular coagulation. Crit Care Med 2005;33:1365–1370.[CrossRef][Medline]
  8. Slofstra SH, Ten Cate H, Spek CA. Signal transduction induced by activated protein C: no role in protection against sepsis? Trends Mol Med 2006;12:374–381.[CrossRef][Medline]
  9. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699–709.[Abstract/Free Full Text]
  10. Dhainaut JF, Marin N, Mignon A, Vinsonneau C. Hepatic response to sepsis: interaction between coagulation and inflammatory processes. Crit Care Med 2001;29:S42–S47.[CrossRef][Medline]
  11. Cerra FB. Hypermetabolism, organ failure, and metabolic support. Surgery 1987;101:1–14.[Medline]
  12. Baue AE. Multiple, progressive, or sequential systems failure: a syndrome of the 1970s. Arch Surg 1975;110:779–781.[Medline]
  13. Pine RW, Wertz MJ, Lennard ES, Dellinger EP, Carrico CJ, Minshew BH. Determinants of organ malfunction or death in patients with intra-abdominal sepsis: a discriminant analysis. Arch Surg 1983;118:242–249.[Abstract]
  14. Slofstra SH, Ten Cate H, Spek CA. Low dose endotoxin priming is accountable for coagulation abnormalities and organ damage observed in the Shwartzman reaction: a comparison between a single-dose endotoxemia model and a double-hit endotoxin-induced Shwartzman reaction. Thromb J 2006;4:13.[CrossRef][Medline]
  15. Brozna JP. Shwartzman reaction. Semin Thromb Hemost 1990;16:326–332.[Medline]
  16. Garrison RN, Spain DA, Wilson MA, Keelen PA, Harris PD. Microvascular changes explain the "two-hit" theory of multiple organ failure. Ann Surg 1998;227:851–860.[CrossRef][Medline]
  17. Slofstra SH, Spek CA, ten Cate H. Disseminated intravascular coagulation. Hematol J 2003;4:295–302.[CrossRef][Medline]
  18. Slofstra SH, Groot AP, Reitsma PH, ten Cate H, Spek CA. Microarray analysis points towards C/EBPd as a novel mediator in endotoxin-induced DIC. Brit J Haematology 2006;133(Suppl 1):31–32.
  19. Archer L. Pathologic manifestations of septic shock. In: Proctor RA, editor. Handbook of endotoxin. Amsterdam, Elsevier Science; 1986. pp. 18–54.
  20. Leemans JC, Stokman G, Claessen N, Rouschop KM, Teske GJ, Kirschning CJ, Akira S, van der Poll T, Weening JJ, Florquin S. Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney. J Clin Invest 2005;115:2894–2903.[CrossRef][Medline]
  21. Poli V. The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J Biol Chem 1998;273:29279–29282.[Free Full Text]
  22. Takahashi H, Tashiro T, Miyazaki M, Kobayashi M, Pollard RB, Suzuki F. An essential role of macrophage inflammatory protein 1alpha/CCL3 on the expression of host's innate immunities against infectious complications. J Leukoc Biol 2002;72:1190–1197.[Abstract/Free Full Text]
  23. O'Grady NP, Tropea M, Preas HL II, Reda D, Vandivier RW, Banks SM, Suffredini AF. Detection of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta during experimental endotoxemia and human sepsis. J Infect Dis 1999;179:136–141.[CrossRef][Medline]
  24. Hermans PW, Hazelzet JA. Plasminogen activator inhibitor type 1 gene polymorphism and sepsis. Clin Infect Dis 2005;41:S453–S458.[CrossRef][Medline]
  25. Biemond BJ, Levi M, Ten Cate H, Van der Poll T, Buller HR, Hack CE, Ten Cate JW. Plasminogen activator and plasminogen activator inhibitor I release during experimental endotoxaemia in chimpanzees: effect of interventions in the cytokine and coagulation cascades. Clin Sci (Lond) 1995;88:587–594.[Medline]
  26. Hack CE, Hart M, van Schijndel RJ, Eerenberg AJ, Nuijens JH, Thijs LG, Aarden LA. Interleukin-8 in sepsis: relation to shock and inflammatory mediators. Infect Immun 1992;60:2835–2842.[Abstract/Free Full Text]
  27. Landmann R, Zimmerli W, Sansano S, Link S, Hahn A, Glauser MP, Calandra T. Increased circulating soluble CD14 is associated with high mortality in gram-negative septic shock. J Infect Dis 1995;171:639–644.[Medline]
  28. Antal-Szalmas P. Evaluation of CD14 in host defence. Eur J Clin Invest 2000;30:167–179.[CrossRef][Medline]
  29. Hu X, Yee E, Harlan JM, Wong F, Karsan A. Lipopolysaccharide induces the antiapoptotic molecules, A1 and A20, in microvascular endothelial cells. Blood 1998;92:2759–2765.[Abstract/Free Full Text]
  30. Arvelo MB, Cooper JT, Longo C, Daniel S, Grey ST, Mahiou J, Czismadia E, Abu-Jawdeh G, Ferran C. A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis. Hepatology 2002;35:535–543.[CrossRef]
  31. Zhou Q, Zhao J, Wiedmer T, Sims PJ. Normal hemostasis but defective hematopoietic response to growth factors in mice deficient in phospholipid scramblase 1. Blood 2002;99:4030–4038.[Abstract/Free Full Text]
  32. Duriez C, Moyret-Lalle C, Falette N, El-Ghissassi F, Puisieux A. BTG2, its family and its tutor. Bull Cancer 2004;91:E242–E253.[Medline]
  33. Boiko AD, Porteous S, Razorenova OV, Krivokrysenko VI, Williams BR, Gudkov AV. A systematic search for downstream mediators of tumor suppressor function of p53 reveals a major role of BTG2 in suppression of Ras-induced transformation. Genes Dev 2006;20:236–252.[Abstract/Free Full Text]
  34. Bradley JR, Pober JS. Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene 2001;20:6482–6491.[CrossRef][Medline]
  35. Kanamori M, Suzuki H, Saito R, Muramatsu M, Hayashizaki Y. T2BP, a novel TRAF2 binding protein, can activate NF-kappaB and AP-1 without TNF stimulation. Biochem Biophys Res Commun 2002;290:1108–1113.[CrossRef][Medline]
  36. Collazo CM, Yap GS, Sempowski GD, Lusby KC, Tessarollo L, Woude GF, Sher A, Taylor GA. Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection. J Exp Med 2001;194:181–188.[Abstract/Free Full Text]
  37. Natsuka S, Akira S, Nishio Y, Hashimoto S, Sugita T, Isshiki H, Kishimoto T. Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. Blood 1992;79:460–466.[Abstract/Free Full Text]
  38. Hu HM, Tian Q, Baer M, Spooner CJ, Williams SC, Johnson PF, Schwartz RC. The C/EBP bZIP domain can mediate lipopolysaccharide induction of the proinflammatory cytokines interleukin-6 and monocyte chemoattractant protein-1. J Biol Chem 2000;275:16373–16381.[Abstract/Free Full Text]
  39. Beck GC, Rafat N, Brinkkoetter P, Hanusch C, Schulte J, Haak M, van Ackern K, van der Woude FJ, Yard BA. Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factors. Clin Exp Immunol 2006;143:523–533.[CrossRef][Medline]
  40. Thomas B, Berenbaum F, Humbert L, Bian H, Bereziat G, Crofford L, Olivier JL. Critical role of C/EBPdelta and C/EBPbeta factors in the stimulation of the cyclooxygenase-2 gene transcription by interleukin-1beta in articular chondrocytes. Eur J Biochem 2000;267:6798–6809.[Medline]
  41. Thangaraju M, Rudelius M, Bierie B, Raffeld M, Sharan S, Hennighausen L, Huang AM, Sterneck E. C/EBPdelta is a crucial regulator of pro-apoptotic gene expression during mammary gland involution. Development 2005;132:4675–4685.[Abstract/Free Full Text]
  42. Sterneck E, Paylor R, Jackson-Lewis V, Libbey M, Przedborski S, Tessarollo L, Crawley JN, Johnson PF. Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein delta. Proc Natl Acad Sci USA 1998;95:10908–10913.[Abstract/Free Full Text]
  43. Ratajczak T, Williams PM, DiLorenzo D, Ringold GM. Multiple elements within the glucocorticoid regulatory unit of the rat alpha 1-acid glycoprotein gene are recognition sites for C/EBP. J Biol Chem 1992;267:11111–11119.[Abstract/Free Full Text]
  44. Kawamura N, Singer L, Wetsel RA, Colten HR. Cis- and trans-acting elements required for constitutive and cytokine-regulated expression of the mouse complement C3 gene. Biochem J 1992;283:705–712.[Medline]
  45. Ochrietor JD, Harrison KA, Zahedi K, Mortensen RF. Role of STAT3 and C/EBP in cytokine-dependent expression of the mouse serum amyloid P-component (SAP) and C-reactive protein (CRP) genes. Cytokine 2000;12:888–899.[CrossRef][Medline]
  46. Huang JH, Liao WS. Induction of the mouse serum amyloid A3 gene by cytokines requires both C/EBP family proteins and a novel constitutive nuclear factor. Mol Cell Biol 1994;14:4475–4484.[Abstract/Free Full Text]
  47. Taylor FB Jr. Staging of the pathophysiologic responses of the primate microvasculature to Escherichia coli and endotoxin: examination of the elements of the compensated response and their links to the corresponding uncompensated lethal variants. Crit Care Med 2001;29(Suppl 1):S78–S89.[CrossRef][Medline]
  48. Schoenmakers SH, Versteeg HH, Groot AP, Reitsma PH, Spek CA. Tissue factor haploinsufficiency during endotoxin induced coagulation and inflammation in mice. J Thromb Haemost 2004;2:2185–2193.[CrossRef][Medline]
  49. Mahidhara R, Billiar TR. Apoptosis in sepsis. Crit Care Med 2000;28:N105–N113.[CrossRef][Medline]

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