Toll-like receptors (TLRs) located on immune cells can identify special conservative microbial motifs (pathogens-associated molecular patterns (PAMPs)), activate signal transduction pathways, induce inflammatory response, and promote the activation of antigen-presenting cells such as mononuclear cells.1-4 In many pathophysiological processes, TLRs play a very important role in inflammation and ischemia-reperfusion injury (IRI) as well.4-8 According to reports,7,8 TLR2 and TLR4 are the most important members in the TLRs family which can recognize biologically and non-biologically-derived endogenous stimuli characteristically, activate nuclear factor κB (NF-κB) as well as active protein-1, and amplify the inflammatory response. Besides these, by mediating a variety of transmembrane signal transduction processes, TLR2 and TLR4 can amplify the inflammatory response.9
In the early-stage of liver transplantation, acute lung injury (ALI) is one of the most serious complications which affects turnover of the disease directly.10,11 Matuschak et al12 found that the damage of intestinal mucosa, bacterial translocation and entero-endotoxemia were related to the ischemia-reperfusion injury of graft (liver) and the intestinum during OLT. These accidents can trigger the inflammatory response to the body of the recipient. At present, more and more researches have proved that ALI in the recipient early post-OLT may be due to the uncontrolled partial lung inflammation.13-16
Mononuclear cell is an important inflammatory cell which could migrate to different parts of the body and differentiate into several types of cells such as alveolar macrophages, Kupffer cells, and phagocytic cells.17,18 Some studies have indicated the importance of mononuclear phagocytes in ALI which also induces endotoxin and reperfusion injury.19,20
So far, data are not available to show the relationship between the expression of TLR2 and TLR4 in mononuclear cells and postoperative ALI in OLT recipients. To demonstrate the mechanism of ALI, we investigated the dynamic expression of TLR2/4 in mononuclear cells as well as the level of the serum inflammatory factors, and analyzed the relationship between the expression of TLR2/4 and ALI in recipients after OLT.
This study was approved by the Research Ethics Board of the Third Affiliated Hospital, Sun Yet-sen University, China. Informed consent was obtained from each patient and their relatives. Twenty-seven patients with end-stage liver disease (23 men and 4 women) were selected to undergo modified piggyback liver transplantation. Among them, 12 suffered from hepatitis B cirrhosis, 8 from cirrhotic hepatitis accompanied by small liver cancer, 4 from chronic severe hepatitis, and 3 from acute liver failure. Eleven patients were of Child's grade A, 6 grade B, and 10 grade C. All of the 27 patients belonged to ASA classification III or IV. They were divided into ALI group and non-ALI group according to the diagnostic criteria of ALI within 7 days after OLT.
To avoid analysis bias, patients were not included in this study if they had had such abnormalities before operation as oxygenation index <300, respiratory infection or respiratory failure.
Anesthesia was induced with intravenous fentanyl (5 μg/kg) and propofol (1-2 mg/kg). Tracheal intubation was done with vecuronium (0.1 mg/kg), and mechanical ventilation was performed with 50% O2. End-tidal CO2 (ETCO2) was maintained at 35-45 mmHg. Anesthesia was maintained with isoflurane (end tidal concentration 0.5%-2.5%) and intermittent intravenous injection of fentanyl 50 to 100 μg. After induction of anesthesia, any hypotension was treated with fluids and dopamine infusion (2 to 6 μg-1min-1). The body temperature was kept at 36-37° C by convectional warming. The autologous blood recovery system was not used in all patients during the operation. To avoid interference of measurements of TLR2/4 expression in mononuclear cells, no whole blood was transfused.
OLT was divided into anhepatic phase (from vascular clamping to reperfusion of the portal vein and inferior vena cava) and neohepatic phase (from reperfusion of donor liver to the end of operation).
The general data of every patient were collected including Child-Turcotte-Pugh (CTP) scores, ASA classification, operative duration, volume of blood loss, and input. These data were analyzed according to the diagnostic criteria of ALI within 7 days after OLT.
Collection of blood samples
Central venous blood (4 ml) of the patients were collected at T1 (after induction of anesthesia), T2 (25 minutes after anhepatic phase), T3 (3 hours after graft reperfusion), and T4 (24 hours after graft reperfusion). Two milliliters of blood was used to measure the expression of TLR2/4 in mononuclear cells by a flow cytometer and the remaining 2 ml was used to measure the serum concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-8 by enzyme linked immuno-sorbent assay (ELISA).
Analysis of TLR2/4 expression
All monoclonal antibodies used in this study were bought from American eBioscience Company (San Diego, California, USA). Totally 20 μl fluoresceinisothio-cyanate (FITC) anti-human TLR 2, phycoerythrin (PE) anti-human TLR 4, and allophycocyanin (APC) anti-human CD 14 were added into 100 μl whole blood treated by EDTA respectively, then mixed thoroughly. The blood mixtures were stained for 20 minutes in the dark at ambient temperature. They were then mixed with 2 ml of red blood cell (RBC) lysis buffer (eBioscience Co.), away from light for 15 minutes and then centrifuged for 5 minutes at 1000 r/min. After rinsing twice, the supernatant was discarded and samples were preserved at 4°C away from light. The negative control samples were incubated with FITC mouse IgG2α and PE mouse IgG2α and run through the entire procedures. APC-CD14 positive cells (monocytes) were chosen and the TLR2/4 expression percentage of 5000 monocytes was determined by Becton Dickinson FACsCalibur flow cytometry (BD, Franklin Lakes, New Jersey, USA).
Assay of inflammatory factors
TNF-α, IL-1β and IL-8 were measured by ELISA kit according to the manufacturer's instructions (Rapidbio Inc., West Hills, California, USA).
Occurrence of ALI within 7 days after OLT
Through follow-up visit and arterial blood gas analysis, we observed the occurrence of ALI within 7 days after the operation and analyzed the relationship between ALI and TLR2/4 expression in monocytes. The diagnostic criteria21 for ALI were acute onset of lung inflammation, PaO2/FiO2 (P/F) ≤300, and nomotopia sternite showing severe infiltration in both lungs with pulmonary artery wedge pressure ≤18 mmHg or without clinical evidence of congestive cardiac failure.
The data were expressed as mean±standard deviation. General linear model for repeated measurement was used to analyze the difference between the phases in the same group. Independent-samples t test was performed to analyze the difference between the two groups. Data of non-normal distribution were expressed as median (interquartile range), and the difference between the two phases in the same group was determined by Wilcoxon's signed-rank test and Spearman's rank-order correlation coefficient. The relationship between TLR2/4 expression in monocytes and ALI was analyzed by Fisher's exact test. Differences at P <0.05 were considered statistically significant. All data were processed by SPSS 12.0 for windows (SPSS Inc., Chicago, Illinois, USA).
Status of postoperative ALI
The 27 patients were operated on successfully. One patient died after OLT (ALI group).
Follow-up visit and arterial blood gas analysis showed that oxygenation index in 14 patients were lower than 300 within 7 days after OLT, and 9 of them were diagnosed with ALI. The mean time of ALI occurrence was 17.7 hours (3-56 hours). There was a positive correlation between time of up-regulation of TLR2 expression and time of ALI occurrence after OLT (r=0.844, P=0.004).
Twenty-seven patients were divided into ALI group (n=9) and non-ALI group (n=18). In the ALI group, 4 patients were of Child's grade A, 2 grade B, and 3 grade C; in the non-ALI group, 7 patients were of Child's grade A, 4 grade B, and 7 grade C. There was no significant difference in CTP scores, age, gender and body weight between the ALI group and non-ALI group (P >0.05).
General data during OLT
Compared to the non-ALI group, the volumes of blood loss, ascites, total blood loss and transfused RBC were higher in the ALI group, and the anhepatic phase also lasted longer (Table 1).
TLR2/4 expression in mononuclear cells in peri-OLT
Compared to T1, the TLR2/4 expression increased in the beginning of anhepatic phase but it was not statistically different at T2 (P >0.05). However it was increased significantly at T3 (P <0.05) and T4 (P <0.01). Multiple comparison analysis showed that the TLR2/4 expression at T3 and T4 was higher than that at T2 (P <0.01), but there was no significant difference between the expression of TLR2/4 at T3 and that at T4 (P >0.05) (Table 2).
Difference of TLR2/4 expression in mononuclear cells between the ALI group and non-ALI group during peri-OLT
In comparison with the non-ALI group, the TLR2/4 expression in mononuclear cells during OLT was significantly increased in the ALI group. In the non-ALI group, the expression level of TLR2/4 in mononuclear cells was not significantly different during peri-OLT, except TLR4 expression at T4 (Table 2).
We calculated the magnitude of change of TLR2/4 according to the following formula: Δ(%) =((Tx—T1) /T1)×100% (T1: the expression of TLR2/4 at T1; Tx: the expression of TLR2/4 at T2, T3 or T4). The data of analysis showed that increased amplitude of TLR2/4 expression during peri-OLT in the ALI group was higher than that in the non-ALI group (Table 3).
Serum levels of TNF-α, IL-1β and IL-8 during peri-OLT
Compared with T1, the serum concentration of TNF-α, IL-1β and IL-8 during peri-OLT increased gradually. There was a statistical difference at T3 and T4 (Table 4).
The serum levels of TNF-α, IL-1β and IL-8 were not significantly different in the two groups during peri-OLT. In the ALI group, the increasing serum levels of TNF-α, IL-1β and IL-8 were significant (Table 4).
The serum concentrations of TNF-α and IL-1β were positively correlated to the expression of TLR4 in mononuclear cells in the ALI group (TNF-α: r=0.342, P=0.041; IL-1β: r=0.335, P=0.046). But no correlation was observed between the concentration of serum inflammatory factors and the expression of TLR2/4 in mononuclear cells in the non-ALI group.
The TLR2/4 expression was increased one fold at T1 in 14 patients, of whom 8 had ALI after OLT. Fisher's exact test revealed that patients with this status were highly possible to have ALI (P=0.013), with a relative risk of 16 (Table 5).
ALI as a widespread complication seriously threatens the lives of liver recipients and their quality of life after liver transplantion.22 Acorrding to Aduen and his co-workers,11 the incidence rate of ALI is about 40%. In the present study, the oxygenation index was lower than 300 within 7 days after OLT in 14 patients, 9 of them met the diagnostic criteria of ALI. One patient died from pulmonary infection because the weaning from mechanical ventilation is diffucult for a long time. After exquisite calculation, the incidence rate of pulmonary infection in OLT patients is 33.3%. At the same time, we found that the volume of blood loss, blood transfusion (RBC) and output, ascites, and anhepatic duration in the ALI group were higher than those in the non-ALI group, indicating that there was a potential positive correlation between the incidence rate of pulmonary infection in OLT recipients, degree of trauma, and duration of intestinal hemostasis and hypoxia.
Fu et al23 confirmed that lipopolysaccharide (LPS) induced the expression of the TLR4 gene in granulocytes and endotheliocytes with up-regulation and activation of NF-κB as well as the production of TNF-α, IL-6 and IL-8 in cultured cells. These effects of LPS were apparently inhibited by TLR4 specific antibody. Shimamoto et al reported that the high expression of TLR4 was positively correlated with ischemia-reperfusion injury. Amstrong et al25 demonstrated that TLR2 mRNA was up-regulated significantly in monocytes in patients with systemic inflammatory response syndrome. This was consistent with the result of detection of TLR2 protein. All of the researches emphasized that TLR2/4 indubitably act as a critical element in inflammatory reaction. Coincidently, in this study the expression of TLR2/4 in mononuclear cells at 3 and 24 hours after graft reperfusion increased significantly along with the increase of serum levels of TNF-α, IL-1β and IL-8. During peri-OLT, systemic inflammatory reaction indeed existed with the up-regulation of TLR2/4 expression in mononuclear cells.
Many studies have proved that under the systematic inflammatory status, serum inflammatory factors of ALI patients increased significantly, a sign of uncontrolled pneumono-injuries.13-16,26-28 In this study the serum levels of TNF-α, IL-1β and IL-8 increased in the ALI group and non-ALI group. Hence we conclude that patients who have undergone OLT may suffer from systemic inflammatory response syndrome after OLT. By comparison, a positive correlation was found between the concentrations of serum TNF-α, IL-1β and the expression of TLR2/4 in mononuclear cells in the ALI group. Moreover, the time for up-regulation of TLR2 expression was also positively correlated with the occurrence of ALI after OLT. These results showed that perioperative inflammatory reaction of OLT and postoperative ALI are relevant to up-regulation of TLR2/4 expression.
In contrast to the non-ALI group, TLR2/4 expression in mononuclear cells during peri-OLT increased significantly in the ALI group at 3 and 24 hours, whereas the magnitude of change of TLR2/4 expression (up-regulation) in the ALI group was increased one fold as in the non-ALI group. In the 14 patients who had this status, 8 suffered from ALI after OLT. Fisher's exact test revealed that the patients whose TLR2/4 expression in mononuclear cells exceeded that at T1 by one fold were highly possible to suffer from ALI (P=0.013) with a relative risk of 16.
In conclusion, expanded injuries and anhepatic phase elongation may contribute to the progression of ALI at early stage, and the high expression of TLR2/4 in mononuclear cells may be an important mechanism for ALI after OLT. Attention should be paid to those recipients during the operative period. Interfering with the expression of TLR2/4 in mononuclear cells may be a new way to prevent and treat ALI after OLT.
1. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the drosophila Toll protein signals activation of adaptive immunity. Nature 1997; 388: 394-397.
2. Arancibia SA, Beltrán CJ, Aguirre IM, Silva P, Peralta AL, Malinarich F, et al. Toll-like receptors are key participants in innate immune responses. Biol Res 2007; 40: 97-112.
3. Gay NJ, Gangloff M, Weber AN. Toll-like receptors as molecular switches. Nat Rev Immunol 2006; 6: 693-698.
4. Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nat Immunol 2004; 5: 975-979.
5. Weighardt H, Holzmann B. Role of Toll-like receptor responses for sepsis pathogenesis. Immunobiology 2007; 212: 715-722.
6. Zacharowski K, Zacharowski PA, Koch A, Baban A, Tran N, Berkels R, et al. Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome. Proc Natl Acad Sci USA. 2006; 103: 6392-6397.
7. Kawai T, Akira S. TLR signaling. Semin Immunol 2007; 19: 24-32.
8. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4: 499-511.
9. Sabroe I, Parker LC, Dower SK, Whyte MK. The role of TLR activation in inflammation. J Pathol 2008; 214: 126-135.
10. Sykes E, Cosgrove JF, Nesbitt ID, O'Suilleabhain CB. Early noncardiogenic pulmonary edema and the use of PEEP and prone ventilation after emergency liver transplantation. Liver Transpl 2007; 13:459-462.
11. Aduen JF, Stapelfeldt WH, Johnson MM, Jolies HI, Grinton SF, Divertie GD, et al. Clinical relevance of time of onset, duration, and type of pulmonary edema after liver transplantation. Liver Transpl 2003; 9: 764-771.
12. Matuschak GM, Henry KA, Johanns CA, Lechner AJ. Liver-lung interactions following Escherichia coli bacteremic sepsis and secondary hepatic ischemia/reperfusion injury. Am J Respir Crit Care Med 2001; 163: 1002-1009.
13. Bauer TT, Montón C, Torres A, Cabello H, Fillela X, Maldonado A, et al. Comparison of systemic cytokine levels in patients with acute respiratory distress syndrome, severe pneumonia and controls. Thorax 2000; 55: 46-52.
14. Abraham E. Neutrophils and acute lung injury
. Crit Care Med 2003; 31(4 Suppl): S195-S199.
15. Jie Z, Cai Y, Yang W, Jin M, Zhu W, Zhu C. Protective effects of alpha 1-antitrypsin on acute lung injury
in rabbits induced by endotoxin. Chin Med J 2003; 116: 1678-1682.
16. Faybik P, Hetz H, Krenn CG, Baker A, Berlakovich GA, Steltzer H. Perioperative cytokines during orthotopic liver transplantation without venovenous bypass. Transplant Proc 2003; 35: 3019-3021.
17. Lorenz E, Patel DD, Hartung T, Schwartz DA. Toll-like receptor 4 (TLR4)-deficient murine macrophage cell line as an in vitro assay system to show TLR4-independent signaling of Bacteroides fragilis lipopolysaccharide. Infect Immun 2002; 70: 4892-4896.
18. Speer CP, Gahr M. The monocyte-macrophage system in the human. Monatsschr Kinderheilkd 1989; 137: 390-395.
19. Shenkar R, Coulson WF, Abraham E. Hemorrhage and resuscitation induce alterations in cytokine expression and the development of acute lung injury
. Am J Respir Cell Mol Biol 1994; 10: 290-297.
20. Koch T, Ragaller M, Haufe D, Hofer A, Grosser M, Albrecht DM, et al. Perfluorohexane attenuates proinflammatory and procoagulatory response of activated monocytes and alveolar macrophages. Anesthesiology 2001; 94: 101-109.
21. Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, et al. The American-European Consensus Conference on ARDS: definitions, mechanisms, and relevant outcomes, clinical trial coordination. Am J Respir Crit Care Med 1994; 149: 818-824.
22. Hong SK, Hwang S, Lee SG, Lee LS, Ahn CS, Kim KH, et al. Pulmonary complications following adult liver transplantation. Transplant Proc 2006; 38: 2979-2981.
23. Fu SL, Hsu YH, Lee PY, Hou WC, Hung LC, Lin CH, et al. Dioscorin isolated from Dioscorea alata activates TLR4-signaling pathways and induces cytokine expression in macrophages. Biochem Biophys Res Commun 2006; 339: 137-144.
24. Shimamoto A, Chong AJ, Yada M, Shomura S, Takayama H, Fleisig AJ, et al. Inhibition of toll-like receptor 4 with Eritoran attenuates myocardial ischemia-reperfusion injury. Circulation 2006; 114(1 Suppl): 1270-274.
25. Armstrong L, Medford AR, Hunter KJ, Uppington KM, Millar AB. Differential expression of Toll-like receptor (TLR)-2 and TLR-4 on monocytes in human sepsis. Clin Exp Immunol 2004; 136: 312-319.
26. Sun D, Zhao M, Ma D, Liao S, Di C. Protective effect of interleukin-1 receptor antagonist on oleic acid-induced lung injury. Chin Med J 1996; 109: 522-526.
27. Fein AM, Calalang-Colucci MG. Acute lung injury
and acute respiratory distress syndrome in sepsis and septic shock. Crit Care Clin 2000; 16:289-317.
28. Wen XH, Kong HY, Zhu SM, Xu JH, Huang SQ, Chen QL. Plasma levels of tumor necrotic factor-alpha and interleukin-6, -8 during orthotopic liver transplantation and their relations to postoperative pulmonary complications. Hepatobiliary Pancreat Dis Int 2004; 3: 38-41.