INTRODUCTION
Cardiopulmonary bypass (CPB) is associated with a generalized inflammatory response which, in a small proportion (1%-2%) of patients, may lead to postoperative complications, including the systemic inflammatory response syndrome, multiple organ failure, and a compromised immune response. The mechanisms by which CPB and surgery modulate immune function are not well understood.
Monocytes and neutrophils play an important role in inflammation and the innate immune response to bacterial infection. These cells are activated when Toll-like receptors (TLRs) located on the cell surface recognize bacterial components, including lipopolysaccharide (LPS) and peptidoglycan (PepG). Peptidoglycan is a major constituent of gram-positive bacterial cell walls and exhibits many endotoxic properties in vivo and in vitro (1, 2).
Toll-like receptor 4 has been identified as the signaling receptor for LPS (3, 4). In the serum, LPS binds to LPS binding protein, which catalyses its transfer to CD14 on the surface of monocytes (5), facilitating the association of a CD14-TLR4-MD2 receptor complex and the induction of intracellular signaling (6). Toll-like receptor 2 recognizes a wide variety of microbial components, including PepG from gram-positive bacteria (7). Binding of bacterial cell wall components to TLRs on monocytes leads to the synthesis and release of proinflammatory cytokines and chemokines, including tumor necrosis factor (TNF) α, interleukin (IL) 1β, and IL-8, as well as anti-inflammatory cytokines such as IL-10 (8), which orchestrate the innate and adaptive immune response. In neutrophils, TLR activation leads to changes in adhesion molecule expression, IL-8 production, enhanced phagocytosis, and induction of the respiratory burst (9, 10). Toll-like receptors may also play an important role in the induction of the systemic inflammatory response to CPB in the absence of infection. Not only has translocation of LPS and PepG from the gastrointestinal tract been implicated in the induction of inflammation after CPB (11) but TLR4 has also been shown to be activated by endogenous danger signals such as the heat shock proteins that may also mediate the inflammatory response to CPB (12).
Numerous studies have documented that shortly after commencing CPB, circulating levels of proinflammatory cytokines and chemokines are increased (reviewed in (13)). These mediators are released by both leukocytes and the myocardium (14), and analysis of circulating leukocytes demonstrates a "primed" phenotype, potentially increasing susceptibility to postoperative infectious complications (15). Anti-inflammatory cytokines are also induced (13), potentially limiting the detrimental effects of the proinflammatory response. The balance between pro- and anti-inflammatory cytokine production is thought to be important in determining the consequences of the inflammatory response. Indeed, a number of studies have demonstrated that CPB is also associated with a degree of immune suppression, as indicated by impaired ex vivo cytokine responses to LPS in whole blood (16-22), although reduced ex vivo cytokine production does not seem to correlate with postoperative morbidity (22).
Although a number of investigations have documented the immune dysregulation during and after CPB, the precise mechanisms involved are poorly understood. Furthermore, the effect of CPB on the response to gram-positive bacterial cell wall components has not previously been investigated, although one study has demonstrated suppression of cytokine responses to a Staphylococcus aureus lysate (19). It is known that the immune hyporeactivity associated with the systemic inflammatory response syndrome is not a generalized phenomenon (23), and it is therefore important to understand how CPB may affect the immune response to components of gram-positive bacteria and to mixed bacterial stimuli, particularly in light of the increasing prevalence of gram-positive and mixed infections. The importance of a thorough understanding of the immune paresis after surgery and CPB lies in the potential to manipulate or attenuate the inflammatory response to reduce postoperative morbidity, particularly infective complications, and improve outcome.
The aim of this study was to characterize the time-related changes in ex vivo cytokine responses to LPS or PepG (alone and in combination with LPS) in whole blood from patients undergoing CPB. Additionally, we hypothesized that altered expression of TLRs on monocytes and neutrophils might be involved in the reduced inflammatory response to bacterial cell wall components after CPB.
MATERIALS AND METHODS
Patients
With the approval of the East London and the City Local Research Ethics Committee, and following written informed consent, we recruited 6 patients scheduled to undergo CPB for routine elective coronary artery grafting or single-valve replacement surgery (Table 1). We excluded immunocompromised patients (including those taking systemic steroids), patients with systemic immune disorders, active infection or inflammation, or those taking nonsteroidal anti-inflammatory drugs or aspirin in the preceding 5 days.
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Table 1: Demographics and perioperative data for patients undergoing CPB
Anesthesia and CPB technique
All regular cardiac medications (with the exception of aspirin) were continued until the day of surgery. Patients were premedicated with an oral administration of benzodiazepine (temazepam) on the morning of surgery. Electrocardiogram, arterial, and central venous pressure, oxygen saturation, inspired and end-tidal gas concentrations, and temperature (nasopharyngeal probe) were monitored continuously perioperatively. Anesthesia was standardized and induced with fentanyl, thiopentone, and rocuronium. After tracheal intubation, patients were mechanically ventilated with oxygen-enriched air (inspired oxygen fraction 0.5) to maintain adequate oxygenation and normocapnia. Anesthesia was maintained with isoflurane and fentanyl before the onset of CPB and with a continuous infusion of propofol during and after CPB. Routine antibiotic prophylaxis was administered.
Initial anticoagulation was achieved with heparin sodium (3 mg/kg) (CP Pharmaceuticals, Wrexham, UK) and supplemented as necessary to maintain the activated clotting time at more than 480 s throughout CPB. The right atrium was cannulated for venous drainage, and the ascending aorta was cannulated for arterial return. The extracorporeal circuit consisted of a Jostra HL20 heart-lung machine (Maquet Ltd, Strathclyde, UK) with an occlusive roller pump, polyvinyl chloride tubing (with silicone tubing in the boot pump), cardiotomy reservoir (with filter), and membrane oxygenator (Cobe CML Duo, Sorin Biomedica UK Ltd, Gloucester, UK; or Capiox, Terumo UK Ltd, Knowsley, UK). The circuit was primed with 2000 mL Hartmann solution containing 150 mg heparin sodium and prebypass filtration of the prime performed using a 0.2-μm filter. Nonpulsatile blood flow was maintained at 2.3 to 2.6 L/min per m2 body surface area, and glyceryl trinitrate, metaraminol, and phentolamine were titrated against the blood pressure as required. Myocardial protection was achieved by antegrade infusion of St Thomas 1 cardioplegia solution diluted in cold blood. Moderate hypothermia (32°C) was achieved by means of a heat exchanger in the bypass circuit. At the conclusion of surgery, heparin was antagonized with protamine sulfate to return the activated clotting time to close to the preoperative value. Patients were transferred to the intensive care unit for routine postoperative care, and all made uneventful recoveries.
Blood sampling
A 35-mL sample of whole blood was obtained from the intra-arterial cannula on 3 occasions: preoperatively at the time of line insertion, before the induction of anesthesia, at the conclusion of CPB, and 20 h postoperatively. White blood cell count and differential leukocyte assessment were performed using an LH 750 hematology analyzer (Beckman Coulter, High Wycombe, Bucks, UK). Blood was anticoagulated with heparin sodium (CP Pharmaceuticals) at 30 IU/mL. Baseline cytokine and flow cytometric analyses were performed within 30 min and the remaining blood prepared for ex vivo stimulation experiments as detailed below.
Bacterial cell wall components
Lipopolysaccharides from Escherichia coli 0127:B8 was purchased from Sigma (Poole, Dorset, UK) and diluted for use in sterile saline (sodium chloride, 0.9% wt/vol; Baxter, Newbury, UK). Peptidoglycan was isolated from S. aureus as previously described (24). The isolated PepG was enzymatically digested, after which the expected reverse-phase-high-pressure liquid chromatography muropeptide profile was obtained with no spurious products. Immediately before use, PepG was dispersed by sonication (3000 Hz, 3 × 10 s) and diluted in sterile saline. The content of LPS in the PepG preparation was analyzed by Limulus amebocyte lysate test (COATEST; Chromogenix, Molndal, Sweden) and was found to be below 2 ng LPS/mg PepG.
Ex vivo whole human blood model
Whole blood was incubated in 10-mL Monovette syringes (Sarstedt, Germany) with gentle agitation at 37°C in the presence of LPS (10 ng/mL), PepG (1 μg/mL), or a combination of the two, as previously described (25, 26). Each experiment was accompanied by a control sample in which only diluent (0.9% saline) was added. At predetermined time intervals, blood was removed from the syringes for analysis of cell surface receptor expression by flow cytometry (time zero) and plasma-separated and stored at −20°C for subsequent measurement of cytokines (0, 3, 6, and 12 h).
Flow cytometry
Aliquots (100 μL) of whole blood were stained with 20 μL of fluorescent-labeled monoclonal antibody or concentration-matched isotype control by incubation on ice for 60 min, protected from light. Red blood cells were then subjected to hypotonic lysis by incubation of samples with fluorescence-activated cell sorter lysing solution (Becton Dickinson, Oxford, UK) for 10 min, and cells were pelleted by centrifugation (1000g for 3 min at 4°C). Cells were washed twice in ice-cold wash buffer (Cell Wash; Becton Dickinson) and resuspended in 1% paraformaldehyde (Cell Fix; Becton Dickinson) then stored protected from light at 4°C until analysis. Antibodies were as follows: fluorescein isothiocyanate (FITC)-conjugated antihuman CD14 [Clone 18D11, isotype immunoglobulin (Ig)G1] and FITC-conjugated IgG1 isotype control (Diatec, Oslo, Norway), phycoerythrin (PE)-conjugated antihuman TLR2 (Clone TL2.1, isotype IgG2a) and PE-conjugated antihuman TLR4 (Clone HTA125, isotype IgG2a) (eBioscience, San Diego, Calif, USA), and PE-conjugated IgG2a isotype control (Cymbus Biotechnology, East Leigh, Hampshire, UK). To enhance identification of the monocyte population for TLR analysis, multicolor flow cytometric analysis was used, double-staining for CD14 and TLR2, CD14 and TLR4, or CD14 and IgG2a-PE isotype control.
Flow cytometry was performed using a single-laser FACScan and CellQuest software (Becton Dickinson). For the analysis of double-stained samples, electronic compensation for fluorochrome spectral overlap was set using appropriate single-stained and unstained samples. Electronic gates were created to define the monocyte and neutrophil populations according to their CD14-staining and light scatter characteristics, and 10,000 monocytes were acquired for each sample. Median fluorescence intensity (MFI) in FL-1 and FL-2 was recorded for each sample, and all samples were performed in duplicate. Median fluorescence intensity values were corrected for nonspecific antibody binding by subtracting the MFI measured for the matched isotype control sample.
Cytokine measurement
Tumor necrosis factor α, IL-1β, IL-8, and IL-10 were measured by enzyme immunoassay according to the manufacturer's protocol (CLB, Amsterdam, The Netherlands), with all standards and samples run in duplicate.
Statistical evaluation
White cell counts are presented as mean and standard deviation. Statistical comparison of 2 groups was performed using two-tailed, paired t test. Cytokine and receptor expression data are presented as median and interquartile range. Statistical comparison of 2 groups was performed using Wilcoxon signed-rank test, whereas comparison of more than 2 groups was performed using Friedman test and Wilcoxon signed-rank test using the SPSS 11.5 software package (Chicago Ill, USA). Differences with P values less than 0.05 were considered to be statistically significant (n = 6 for all experiments).
RESULTS
Patient data, leukocyte counts, and spontaneous cytokine production
Despite a 34% fall in hematocrit (Table 2) reflecting hemodilution, the white cell count is increased after CPB largely because of an increase in neutrophils, which are significantly increased at the end of surgery (P < 0.05) and at 20 h postoperatively (P < 0.01). The monocyte count is significantly reduced at the end of surgery (P < 0.01) but increases to above baseline levels by 20 h postoperatively (P < 0.02). In contrast, the lymphocyte count is significantly reduced 20 h postoperatively (P < 0.01).
Table 2: Perioperative leukocyte counts and hematocrit in patients undergoing CPB
No significant TNF α, IL-1β, or IL-8 was detected in plasma taken from patients at any of the sampling points. Interleukin 10 was detectable in plasma preoperatively in 5 of 6 patients (range, 0-446 pg/mL) in all patients at the end of bypass (90-752 pg/mL) and in 4 of 6 patients 20 h postoperatively (0-247 pg/mL). Interleukin 10 levels showed a tendency to peak at the end of CPB and decrease the following day (data not shown).
Influence of CPB on ex vivo cytokine responses to bacterial cell wall components
Tumor necrosis factor α-
The TNF-α responses to ex vivo stimulation with LPS, PepG, or a combination of the 2 were reduced by more than 85% at the end of CPB in comparison with the responses observed in blood taken before the induction of anesthesia (P < 0.03) (Fig. 1A). There was partial (less than 50%) recovery of the TNF-α response to LPS alone and LPS + PepG 20 h later, but the response remained significantly less than that observed preoperatively (P < 0.05). However, there was recovery of the response to PepG alone. At all 3 time points, the TNF-α response to LPS + PepG was significantly greater than the response to either component alone (P < 0.03) and greater than the sum of the individual responses.
Fig. 1: Tumor necrosis factor α (A), IL-1β (B), IL-8 (C), and IL-10 (D) release in whole blood in response to ex vivo stimulation before and after CPB. Whole blood was obtained from patients before the induction of anesthesia, at the end of CPB, and 20 h later. The blood was incubated at 37°C in the presence of LPS (10 ng/mL) (L), PepG (1 μg/mL) (P), a combination of LPS and PepG (L + P), or diluent only (0.9% saline) (Nil). Blood was removed at 3 (white bars), 6 (light gray), and 12 (dark gray) h and plasma-separated by centrifugation. Plasma cytokine levels were measured by enzyme immunoassay. Data are presented as medians and interquartile ranges of values from 6 patients. *P < 0.05 compared with preanesthesia cytokine response.
Interleukin 1β-
The IL-1β responses to ex vivo stimulation with LPS, PepG, or a combination of the two were reduced by more than 90% at the end of CPB in comparison with the responses observed in blood taken before the induction of anesthesia (P < 0.05) (Fig. 1B). There was partial (less than 30%) recovery of the IL-1β response to LPS alone and LPS + PepG 20 h later, but the response remained significantly less than that observed preoperatively (P < 0.05). However, there was full recovery of the response to PepG alone.
Interleukin 8-
The IL-8 responses to ex vivo stimulation with LPS, PepG, or a combination of the two were reduced by more than 70% at the end of CPB in comparison with the responses observed in blood taken before the induction of anesthesia (P < 0.03) (Fig. 1C). There was recovery of the IL-8 response to stimulation with LPS, PepG, and their combination 20 h later.
Interleukin 10-
The IL-10 response to ex vivo stimulation was delayed with levels beginning to rise only after 6 h of exposure to cell wall components. At the conclusion of CPB, the IL-10 responses to ex vivo stimulation with LPS or its combination with PepG were reduced by more than 75% (P < 0.05) (Fig. 1D). The response to PepG after 6 h stimulation was unaffected but was reduced by more than 95% after 12 h stimulation (P < 0.03). There was recovery of all IL-10 responses 20 h later with the exception of the response to LPS stimulation for 12 h, which remained reduced (P < 0.05).
Monocyte cell surface expression of CD14, TLR2, and TLR4
There was no significant change in monocyte CD14 expression at the end of CPB or 20 h later (Fig. 2A) when compared with preanesthesia levels. Toll-like receptor 2 expression on monocytes was unchanged at the end of CPB but was significantly upregulated (121% increase in expression) 20 h later (P < 0.03; Fig. 2B). There was a small (29%), but statistically significant, reduction in the expression of monocyte TLR4 (Fig. 2C) at the end of CPB (P < 0.05), but TLR4 expression was significantly upregulated (132% increase) 20 h postoperatively (P < 0.03).
Fig. 2: Monocyte surface expression of CD14 (A), TLR2 (B), and TLR4 (C), and neutrophil surface expression of CD14 (D), TLR2 (E), and TLR4 (F) in blood obtained from patients before the induction of anesthesia (T1), at the end of CPB (T2), and 20 h later (T3). Aliquots (100 μL) of whole blood were stained with 20 μL FITC-labeled monoclonal antibody to CD14 and 20 μL PE-labeled monoclonal antibody to TLR2 or TLR4 (see "Materials and methods"). Samples were processed by flow cytometry and analyzed using CellQuest software. The MFI for each sample was corrected for nonspecific binding by subtracting the MFI for the concentration-matched isotype control. Data are presented as medians and interquartile ranges of values from 6 patients. †P < 0.05 compared with preanesthesia levels of expression.
Neutrophil surface expression of CD14, TLR2, and TLR4
The expression of CD14 on the cell surface of neutrophils was significantly upregulated (269% increase) at the end of CPB when compared with preanesthesia levels (P < 0.03) (Fig. 2D). Both TLR2 (Fig. 2E) and TLR4 (Fig. 2F) were downregulated on neutrophils at the end of CPB (52% and 53%, respectively; P < 0.03), but expression had returned to baseline preanesthesia levels 20 h later.
DISCUSSION
The present study demonstrates for the first time that after cardiac surgery with CPB, the ability of leukocytes to produce cytokines in response to PepG ex vivo is almost abolished, and that the cytokine response to PepG is recovered 20 h postoperatively (in contrast to the partial recovery of LPS responses). We also demonstrate for the first time that neutrophil TLR2 and TLR4 expression is reduced after CPB, whereas neutrophil CD14 expression is increased. Our findings also indicate that suppression of cytokine responses at the end of CPB is not mediated by downregulation of leukocyte TLR expression, but that upregulation of monocyte TLR expression may partly account for the subsequent recovery of TNF-α, IL-1β, and IL-10 production.
The use of an ex vivo, undiluted whole human blood model to characterize the cellular responses to stimulation with bacterial cell wall components permits ongoing interaction between cells and mediators involved in the innate immune response and avoids stimulating white cell populations by the process of separation from whole blood. The model also permits observation of the contributions of all the leukocyte populations, although the exact cellular origin of the cytokines is not determined. Using this model, we observed 70% to 90% suppression of the ex vivo cytokine responses to bacterial cell wall components at the end of CPB, which is disproportionate to the 40% reduction in circulating monocytes at this time, suggesting a functional suppression of monocyte cytokine release. Tumor necrosis factor α and IL-1β responses to LPS were significantly reduced at the end of CPB, with only partial restoration of responsiveness the following day (when there is a relative monocytosis). This concurs with the findings of other groups using a diluted whole blood model in patients undergoing CPB (16, 17, 19), whereas others have reported that there is recovery of the TNF-α and IL-1β response to LPS 24 h after CPB (18, 21). Additionally, we show here that there is significant suppression of the ex vivo TNF-α and IL-1β responses to PepG at the end of CPB, indicating that immune suppression after CPB is not restricted to LPS responsiveness. The proinflammatory response to PepG did, however, recover more rapidly, having returned to baseline at 20 h. Most previous studies have focused on the effect of CPB on the ex vivo response to LPS. However, a blunted TNF-α response to a S. aureus lysate immediately after CPB has been reported by Wilhelm et al. (19), and, interestingly, these investigators also observed more complete recovery of the response to a gram-positive bacterial stimulus at 24 h (95%) than to LPS (74%).
We observed a greater than 70% reduction in the IL-8 response to bacterial cell wall components at the end of CPB. As we have previously observed in blood from healthy volunteers (26), the IL-8 response to PepG was greater than that observed after exposure to LPS and, with prolonged stimulation, was even greater than the response to LPS in combination with PepG. Interleukin 8 is involved in neutrophil chemotaxis and activation, and plays an important role in tissue injury in the acute respiratory distress syndrome and ischemia-reperfusion injury (27). Much of the tissue damage after CPB is thought to be a consequence of the extravascular migration of activated neutrophils facilitated by cytokine-mediated upregulation of adhesion molecules on neutrophils and endothelial cells (14, 28-32). Because both LPS and PepG are detectable in the circulation after cardiac surgery (11, 33), IL-8 released in response to both bacterial components may contribute to the development of organ injury, especially acute respiratory distress syndrome, by recruiting neutrophils into the tissues.
In the present study, the previously observed suppression of IL-10 responses to ex vivo LPS stimulation after CPB (19-21) was also seen after stimulation with PepG alone and in combination with LPS. However, we found that IL-10 responses to ex vivo stimulation return to baseline at 20 h, whereas others have shown a significant increase in the IL-10 response over baseline at this time point (21).
An important characteristic of PepG is its ability to enhance LPS signaling and augment its toxicity (26, 34, 35). This synergistic interaction has been demonstrated in respect of the release of TNF-α, IL-1β, IL-6, and IL-8 in whole human blood (26, 34), and in the degree of shock and organ injury in a rodent model of sepsis (35). There is, however, no evidence of synergy in the release of the anti-inflammatory cytokine IL-10 in response to coadministration of LPS and PepG (26, 34), and this may be permissive for an exaggerated early proinflammatory response. In this study, using blood from cardiac surgical patients, a synergistic response was observed in the release of TNF-α, but not IL-1β or IL-8. The reasons for this differential response, even before the induction of anesthesia, are not clear, but may include the older age group studied, concurrent disease, and medications, particularly statin therapy, which is known to have immunomodulatory effects (36).
To investigate potential mechanisms underlying the suppression of ex vivo cytokine responses to bacterial cell wall components during CPB, we analyzed the changes in expression of leukocyte cell surface receptors involved in bacterial recognition and signal transduction. Although there was a small reduction in monocyte TLR4 expression at the end of CPB, the blunted cytokine responses to stimulation with bacterial cell wall components observed at the end of bypass could not be explained by receptor downregulation on monocytes. This suggests that altered signal transduction downstream of the TLRs is responsible for abrogating the cytokine response after CPB, as is seen in other situations in which microbial tolerance is observed (37). These mechanisms include inhibition of myleoid differentiation primary response gene 88/TLR4 complex and interleukin 1 receptor-associated kinase (IRAK) 1 activation, increased expression/activity of negative regulators of TLR signaling, including IRAK-M, Tollip, suppressor of cytokine signaling 1, T1/ST2, and SH2 inositol 5-phosphatase, and increased expression of alternatively spliced forms of myleoid differentiation primary response gene 88 and IRAK-1, which fail to interact with downstream signaling molecules (37). The increased monocyte TLR2 and TLR4 expression observed 20 h postoperatively may, along with the concurrent monocytosis, contribute to the recovery of cytokine responses observed at this time point. Increased TLR expression at this time may also contribute to the phenomenon of leukocyte priming (15), leading to an excessive inflammatory response to postoperative infectious stimuli. Similar upregulation of TLR expression on monocytes 19 h post-coronary artery bypass graft has been reported by Dybdahl et al. (12). However, in contrast to our findings, these authors reported that TLR4 expression was unchanged at the end of CPB, whereas TLR2 was upregulated (12). Also, in contrast to our findings, several groups have found that monocyte CD14 is downregulated after CPB (12, 38-40), although with some inconsistency to the timing and duration of the downregulation: it has been reported that CD14 is downregulated at the end of surgery, but normalized by the following day (12, 38, 39), whereas another group reported that monocyte CD14 expression reaches its nadir 20 h postoperatively (40).
We show that the expression of TLR2 and TLR4 on the cell surface of circulating neutrophils is downregulated immediately after CPB but returns to baseline 20 h later. Neutrophils are a source of IL-8 in inflammation and sepsis, and this receptor downregulation coincided with a substantial (more than 70%) reduction in the production of IL-8 in response to ex vivo stimulation. However, downregulation of neutrophil TLR expression is unlikely to fully account for the observed suppression of IL-8 production because monocytes are thought to be the principal source of IL-8.
It is, of course, not possible to differentiate the immunomodulatory effects of the extracorporeal circulation from the effects of anesthesia, anticoagulation, surgery, ischemia-reperfusion, and blood transfusion, all of which are also known to modulate immune function. However, circulation of blood through an isolated experimental extracorporeal circuit has been shown to induce hyporesponsiveness to LPS (20). Interestingly, in a study of patients undergoing major gastrointestinal surgery, TLR2 and TLR4 expression were found to be downregulated postoperatively and to remain downregulated for up to a week (41). Additionally, CD14 expression has been shown to be downregulated after thoracic (39) and major gastrointestinal surgery (42), suggesting an effect of anesthesia and surgery rather than a specific effect of CPB.
In conclusion, cardiac surgery with CPB is associated with the rapid development of leukocyte hyporesponsiveness to PepG and LPS, affecting both pro- and anti-inflammatory cytokines, with substantial recovery the following day. Reduced production of cytokines cannot be explained by downregulation of leukocyte TLR expression, although monocyte receptor upregulation may contribute to subsequent recovery of monocyte responsiveness.
ACKNOWLEDGMENTS
The authors thank Elin Sletbakk (Oslo, Norway) for skilled technical assistance with cytokine analyses.
REFERENCES
1. Wang JE, Dahle MK, Yndestad A, Bauer I, McDonald MC, Aukrust P, Foster SJ, Bauer M, Aasen AO, Thiemermann C: Peptidoglycan of
Staphylococcus aureus causes inflammation and organ injury in the rat.
Crit Care Med 32:546-552, 2004.
2. Timmerman CP, Mattsson E, Martinez-Martinez L, De Graaf L, Van Strijp JA, Verbrugh HA, Verhoef J, Fleer A: Induction of release of tumor necrosis factor from human monocytes by staphylococci and staphylococcal peptidoglycans.
Infect Immun 61:4167-4172, 1993.
3. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S: Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product.
J Immunol 162:3749-3752, 1999.
4. Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, et al: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.
Science 282:2085-2088, 1998.
5. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC: CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.
Science 249:1431-1433, 1990.
6. Jiang Q, Akashi S, Miyake K, Petty HR: Lipopolysaccharide induces physical proximity between CD14 and Toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-kappa B.
J Immunol 165:3541-3544, 2000.
7. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ: Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2.
J Biol Chem 274:17406-17409, 1999.
8. Beutler B, Hoebe K, Du X, Ulevitch RJ: How we detect microbes and respond to them: the Toll-like receptors and their transducers.
J Leukoc Biol 74:479-485, 2003.
9. Sabroe I, Prince LR, Jones EC, Horsburgh MJ, Foster SJ, Vogel SN, Dower SK, Whyte MK: Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation of neutrophil activation and life span.
J Immunol 170:5268-5275, 2003.
10. Hayashi F, Means TK, Luster AD: Toll-like receptors stimulate human neutrophil function.
Blood 102:2660-2669, 2003.
11. Rossi M, Sganga G, Mazzone M, Valenza V, Guarneri S, Portale G, Carbone L, Gatta L, Pioli C, Sanguinetti M, et al: Cardiopulmonary bypass in man: role of the intestine in a self-limiting inflammatory response with demonstrable bacterial translocation.
Ann Thorac Surg 77:612-618, 2004.
12. Dybdahl B, Wahba A, Lien E, Flo TH, Waage A, Qureshi N, Sellevold OF, Espevik T, Sundan A: Inflammatory response after open heart surgery: release of heat-shock protein 70 and signaling through Toll-like receptor-4.
Circulation 105:685-690, 2002.
13. Laffey JG, Boylan JF, Cheng DC: The systemic inflammatory response to cardiac surgery: implications for the anesthesiologist.
Anesthesiology 97:215-252, 2002.
14. Zahler S, Massoudy P, Hartl H, Hahnel C, Meisner H, Becker BF: Acute cardiac inflammatory responses to postischemic reperfusion during cardiopulmonary bypass.
Cardiovasc Res 41:722-730, 1999.
15. Tomic V, Russwurm S, Moller E, Claus RA, Blaess M, Brunkhorst F, Bruegel M, Bode K, Bloos F, Wippermann J, et al: Transcriptomic and proteomic patterns of systemic inflammation in on-pump and off-pump coronary artery bypass grafting.
Circulation 112:2912-2920, 2005.
16. Volk T, Dopfmer UR, Schmutzler M, Rimpau S, Schnitzler H, Konertz W, Hoeflich C, Docke WD, Spies CD, Volk HD, et al: Stress induced IL-10 does not seem to be essential for early monocyte deactivation following cardiac surgery.
Cytokine 24:237-243, 2003.
17. Borgermann J, Friedrich I, Flohe S, Spillner J, Majetschak M, Kuss O, Sablotzki A, Feldt T, Reidemeister JC, Schade FU: Tumor necrosis factor-alpha production in whole blood after cardiopulmonary bypass: downregulation caused by circulating cytokine-inhibitory activities.
J Thorac Cardiovasc Surg 124:608-617, 2002.
18. Kleinschmidt S, Wanner GA, Bussmann D, Kremer JP, Ziegenfuss T, Menger MD, Bauer M: Proinflammatory cytokine gene expression in whole blood from patients undergoing coronary artery bypass surgery and its modulation by pentoxifylline.
Shock 9:12-20, 1998.
19. Wilhelm W, Grundmann U, Rensing H, Werth M, Langemeyer J, Stracke C, Dhingra D, Bauer M: Monocyte deactivation in severe human sepsis or following cardiopulmonary bypass.
Shock 17:354-360, 2002.
20. Dehoux MS, Hernot S, Asehnoune K, Boutten A, Paquin S, Lecon-Malas V, Toueg ML, Desmonts JM, Durand G, Philip I: Cardiopulmonary bypass decreases cytokine production in lipopolysaccharide-stimulated whole blood cells: roles of interleukin-10 and the extracorporeal circuit.
Crit Care Med 28:1721-1727, 2000.
21. Grundmann U, Rensing H, Adams HA, Falk S, Wendler O, Ebinger N, Bauer M: Endotoxin desensitization of human mononuclear cells after cardiopulmonary bypass: role of humoral factors.
Anesthesiology 93:359-369, 2000.
22. Schumacher K, Korr S, Vazquez-Jimenez JF, von Bernuth G, Duchateau J, Seghaye MC: Does cardiac surgery in newborn infants compromise blood cell reactivity to endotoxin?
Crit Care 9:R549-R555, 2005.
23. Cavaillon JM, Adib-Conquy M, Cloez-Tayarani I, Fitting C: Immunodepression in sepsis and SIRS assessed by ex vivo cytokine production is not a generalized phenomenon: a review.
J Endotoxin Res 7:85-93, 2001.
24. Foster SJ: Analysis of the autolysins of
Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis.
J Bacteriol 174:464-470, 1992.
25. Wang JE, Jorgensen PF, Almlof M, Thiemermann C, Foster SJ, Aasen AO, Solberg R: Peptidoglycan and lipoteichoic acid from
Staphylococcus aureus induce tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-10 production in both T cells and monocytes in a human whole blood model.
Infect Immun 68:3965-3970, 2000.
26. Hadley JS, Wang JE, Foster SJ, Thiemermann C, Hinds CJ: Peptidoglycan of
Staphylococcus aureus upregulates monocyte expression of CD14, Toll-like receptor 2 (TLR2), and TLR4 in human blood: possible implications for priming of lipopolysaccharide signaling.
Infect Immun 73:7613-7619, 2005.
27. Sekido N, Mukaida N, Harada A, Nakanishi I, Watanabe Y, Matsushima K: Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8.
Nature 365:654-657, 1993.
28. Nieman G, Searles B, Carney D, McCann U, Schiller H, Lutz C, Finck C, Gatto LA, Hodell M, Picone A: Systemic inflammation induced by cardiopulmonary bypass: a review of pathogenesis and treatment.
J Extra Corpor Technol 31:202-210, 1999.
29. Carney DE, Lutz CJ, Picone AL, Gatto LA, Schiller HJ, Finck CM, Searles B, Paskanik AM, Snyder KP, Edwards C, et al: Soluble tumor necrosis factor receptor prevents post-pump syndrome.
J Surg Res 83:113-121, 1999.
30. Jordan JE, Zhao ZQ, Vinten-Johansen J: The role of neutrophils in myocardial ischemia-reperfusion injury.
Cardiovasc Res 43:860-878, 1999.
31. Gillinov AM, Redmond JM, Zehr KJ, Wilson IC, Curtis WE, Bator JM, Burch RM, Reitz BA, Baumgartner WA, Herskowitz A: Inhibition of neutrophil adhesion during cardiopulmonary bypass.
Ann Thorac Surg 57:126-133, 1994.
32. Byrne JG, Smith WJ, Murphy MP, Couper GS, Appleyard RF, Cohn LH: Complete prevention of myocardial stunning, contracture, low-reflow, and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion.
J Thorac Cardiovasc Surg 104:1589-1596, 1992.
33. Tsunooka N, Maeyama K, Hamada Y, Imagawa H, Takano S, Watanabe Y, Kawachi K: Bacterial translocation secondary to small intestinal mucosal ischemia during cardiopulmonary bypass. Measurement by diamine oxidase and peptidoglycan.
Eur J Cardiothorac Surg 25:275-280, 2004.
34. Wang JE, Jorgensen PF, Ellingsen E, Almlof M, Thiemermann C, Foster SJ, Aasen AO, Solberg R: Peptidoglycan primes for LPS-induced release of proinflammatory cytokines in whole human blood.
Shock 16:178-182, 2001.
35. Wray GM, Foster SJ, Hinds CJ, Thiemermann C: A cell wall component from pathogenic and non-pathogenic gram-positive bacteria (peptidoglycan) synergises with endotoxin to cause the release of tumour necrosis factor-alpha, nitric oxide production, shock, and multiple organ injury/dysfunction in the rat.
Shock 15:135-142, 2001.
36. Blanco-Colio LM, Tunon J, Martin-Ventura JL, Egido J: Anti-inflammatory and immunomodulatory effects of statins.
Kidney Int 63:12-23, 2003.
37. Medvedev AE, Sabroe I, Hasday JD, Vogel SN: Tolerance to microbial TLR ligands: molecular mechanisms and relevance to disease.
J Endotoxin Res 12:133-150, 2006.
38. Sbrana S, Parri MS, De Filippis R, Gianetti J, Clerico A: Monitoring of monocyte functional state after extracorporeal circulation: a flow cytometry study.
Cytometry 58B:17-24, 2004.
39. Hiesmayr MJ, Spittler A, Lassnigg A, Berger R, Laufer G, Kocher A, Artemiou O, Boltz-Nitulescu G, Roth E: Alterations in the number of circulating leucocytes, phenotype of monocyte and cytokine production in patients undergoing cardiothoracic surgery.
Clin Exp Immunol 115:315-323, 1999.
40. Fingerle-Rowson G, Auers J, Kreuzer E, Labeta M, Schmidt B, Samtleben W, Ziegler-Heitbrock HW, Blumenstein M: Down-regulation of surface monocyte lipopolysaccharide-receptor CD14 in patients on cardiopulmonary bypass undergoing aorta-coronary bypass operation.
J Thorac Cardiovasc Surg 115:1172-1178, 1998.
41. Ikushima H, Nishida T, Takeda K, Ito T, Yasuda T, Yano M, Akira S, Matsuda H: Expression of Toll-like receptors 2 and 4 is downregulated after operation.
Surgery 135:376-385, 2004.
42. Kawasaki T, Ogata M, Kawasaki C, Tomihisa T, Okamoto K, Shigematsu A: Surgical stress induces endotoxin hyporesponsiveness and an early decrease of monocyte mCD14 and HLA-DR expression during surgery.
Anesth Analg 92:1322-1326, 2001.
