help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on November 6, 2003, doi:10.1164/rccm.200303-311OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200303-311OCv1
169/3/413    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sewnath, M. E.
Right arrow Articles by Gouma, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sewnath, M. E.
Right arrow Articles by Gouma, D. J.
American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 413-420, (2004)
© 2004 American Thoracic Society

Cholestatic Interleukin-6–Deficient Mice Succumb to Endotoxin-induced Liver Injury and Pulmonary Inflammation

Miguel E. Sewnath, Tom van der Poll, Cornelis J. F. van Noorden, Fiebo J. W. ten Kate and Dirk J. Gouma

Departments of Surgery, Experimental Internal Medicine, Cell Biology and Histology, and Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Correspondence and requests for reprints should be addressed to Miguel E. Sewnath, M.D., P.O. Box 22700, G4-Suite 126, Department of Surgery, Academic Medical Center, 1100 DE Amsterdam, The Netherlands. E-mail: m.e.sewnath{at}olvg.nl


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Circulating and hepatic interleukin (IL)-6 levels are strongly increased during clinical and experimental cholestasis. Cholestatic liver injury is associated with increased susceptibility to endotoxin-induced toxicity. To determine the role of IL-6 herein, extrahepatic cholestasis was induced by bile duct ligation (BDL) in IL-6-gene deficient (IL-6-/-) and normal (IL-6+/+) mice. BDL elicited increased levels of hepatic IL-6 mRNA and protein in normal mice. Hepatocellular injury 2 weeks after BDL was similar in IL-6-/- and IL-6+/+ mice as demonstrated by clinical chemistry and histopathology. Administration of endotoxin to cholestatic mice 2 weeks after BDL was associated with enhanced cytokine release, severe liver damage, and death when compared with sham-operated mice. Effects of endotoxin were largely similar in sham-operated IL-6-/- and IL-6+/+ mice, but cholestatic IL-6-/- mice were more susceptible to the toxic effects of endotoxin, as reflected by increased cytokine release, more profound liver injury and lung inflammation, and higher mortality. Although endogenous IL-6 is not important in the development of liver injury after experimentally induced obstructive jaundice, this cytokine plays an important role in decreasing hypersensitivity to endotoxin in cholestatic mice.

Key Words: acute respiratory distress syndrome • bile duct obstruction • cytokines • knockout mice • sepsis

Cholestatic liver disease is associated with high perioperative morbidity and mortality (1, 2), and is linked with an increased occurrence of pulmonary inflammation and multiple organ failure (3, 4). A factor that has been implicated in the increased risk of perioperative complications during cholestasis is the enhanced sensitivity to the toxic effects of endotoxin, the proinflammatory moiety of the outer membrane of gram-negative bacteria, which accompanies obstructive jaundice (5, 6). During cholestasis, endotoxemia is reported in up to 50–70% of patients (5, 7), often related to the bilirubin content of the blood (8), and the presence of endotoxin in the bloodstream is an important risk factor for patients undergoing surgery for obstructive jaundice (5, 6). Endotoxin is a potent inducer of many inflammatory cascades, including the cytokine network. Previous studies have documented exaggerated release of several cytokines after endotoxin administration to cholestatic animals, including tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-1, and IL-6 (9, 10). This altered cytokine response to a given dose of endotoxin likely plays a role in the eventual effect of this bacterial component on organ damage and lethality. Indeed, we established that the enhanced release of IL-1 in response to endotoxin plays a major part in liver injury and mortality in cholestatic mice (11).

IL-6 is a pleiotrophic cytokine stimulating a variety of cell types, including hematopoietic and neuronal cells, hepatocytes, and nonoval biliary epithelial cells (12, 13). IL-6 also modulates the cytokine levels and hepatic expression of acute-phase response genes during inflammation (14). Hepatic and circulating concentrations of IL-6 are profoundly elevated during experimental and clinical cholestasis (15, 16). Knowledge of the role of this endogenously produced IL-6 in cholestatic liver injury and the ensuing enhanced susceptibility to endotoxin is limited. Therefore, in the present study we used mice with a targeted disruption of their IL-6 gene (IL-6-/- mice) to determine the role of IL-6 in hepatic and lung injury, and mortality during extrahepatic cholestasis induced by bile duct ligation (BDL) in the presence or absence of endotoxemia.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male IL-6-/- mice on a BALB/c background (body weight, 16–20 g) were kindly provided by M. Kopf (Basel Institute for Immunology, Basel, Switzerland). BALB/c IL-6+/+ mice were purchased from Harlan Sprague Dawley (Horst, The Netherlands).

Surgical Procedures
The Institutional Animal Care and Use Committee of the Academic Medical Center (Amsterdam, The Netherlands) approved all experiments. BDL and sham surgeries were performed exactly as described previously (see online supplement for details) (11). In total, 184 IL-6+/+ mice and 184 IL-6-/- mice were used.

Materials
Endotoxin (lipopolysaccharide, Escherichia coli serotype O111:B4) was purchased as a lyophilized powder from Sigma (St. Louis, MO), resuspended in 0.5 ml of sterile pyrogen-free isotonic saline, and injected intraperitoneally (4 µg/kg body weight) 2 weeks after BDL or sham surgery. Control mice received sterile pyrogen-free isotonic saline.

Blood, Bronchoalveolar Lavage Fluid, and Organ Sampling
Mice were killed 1 or 2 weeks after operation, and 1.5, 3, 6, and 24 hours after endotoxin challenge. Blood, bronchoalveolar lavage fluid (BALF), and organ sampling and processing were performed as described previously (see online supplement for details) (11, 17, 18).

Histology and Immunohistochemistry
Histologic examination of paraffin-embedded lung and liver tissue (hematoxylin and eosin) was performed on coded samples by two independent investigators, blinded for treatment groups and biochemical and histochemical parameters. For endotoxin-induced liver injury, two parameters were scored: the area of necrosis and hepatic neutrophil sequestration. The latter were stained by the chloroacetate esterase technique (19) and quantitated as described previously (see online supplement for details) (11).

For lung tissue damage, four histopathologic features were scored: normal, edema, hemorrhage, and severe hemorrhage. Samples were quantitatively scored by level of severity (Grades 0–3), according to previously described methods (see online supplement for details) (20, 21).

Granulocyte staining in lung tissue was performed with fluorescein isothiocyanate-labeled anti-mouse Ly-6-G monoclonal antibody (BD Biosciences Pharmingen, San Diego, CA) exactly as described previously (see online supplement for details) (22).

Cell Counts
Total cells present in BALF were counted in a hematometer, and leukocyte differentiation was determined on Cytospin preparations stained with modified Giemsa stain (DiffQuick products; Dade, Düdingen, Switzerland).

Edema Assessment
Liver and pulmonary edema was evaluated by measuring the wet-to-dry weight ratio as described previously (see online supplement for details) (23).

Assays
Levels of total plasma bilirubin, alkaline phosphatase, {gamma}-glutamyltransferase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined with commercially available kits (Sigma), using a Hitachi analyzer (Roche, Mannheim, Germany) according to the manufacturer's instructions. Cytokines were measured in duplicate by ELISA according to the manufacturer's instructions, with limits of detection (pg/ml) in brackets: TNF-{alpha} [31.3], IL-1{alpha} [8], IL-1ß [8] (all from R&D Systems, Abingdon, UK), and IL-6 [9.8] (BD Biosciences Pharmingen). The myeloperoxidase assay was performed as described previously (see online supplement for details) (24).

RNA Preparation and Reverse Transcriptase-Polymerase Chain Reaction
RNA preparation and reverse transcriptase-polymerase chain reaction (RT-PCR) of liver tissue samples were performed as described previously (see online supplement for details) (11). Cycling conditions for PCR amplification were as follows: 94°C for 5 minutes (1 cycle) followed by 33 (IL-6) or 24 (ß-actin) cycles of 95°C for 60 seconds, 58°C for 60 seconds, and 72°C for 60 seconds, followed by a final extension phase at 72°C for 10 minutes. The primers used were as follows: IL-6 (forward, 5'-CTGGTGACAACCACGGCCTCCCCT-3'; reverse, 5'-ATGCTTAGGCATAACGCACTAGGT-3') and ß-actin (forward, 5'-GTCAGAAGGACTCCTATGTG-3'; reverse, 5'-GCTCGTTGCCAATAGTGATG-3').

Statistical Analysis
Statistics were performed with the SPSS Base 11.0 Statistical Software Package (SPSS, Chicago, IL). All results are given as means ± SE. Survival curves were compared by log-rank test. Differences in biochemical, immunologic, immunohistochemical, and histologic data were analyzed by Mann–Whitney U test. Changes in time were analyzed by one-way analysis of variance. Differences at the indicated time points were assessed by Bonferroni test for multiple comparisons when appropriate. A two-tailed p value < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of IL-6 in Cholestatic Liver Injury
RT-PCR was performed on liver homogenates to determine whether IL-6 is produced after BDL. IL-6 mRNA was detectable in livers of IL-6+/+ mice after induction of extrahepatic cholestasis and in sham-operated animals (Figure 1A) . However, mRNA levels were distinctly higher in the BDL group. Similarly, IL-6 protein levels were higher in liver and plasma of BDL IL-6+/+ mice (Figures 1B and 1C). IL-6 mRNA and protein were never detected in plasma and livers of IL-6-/- mice.




View larger version (89K):
[in this window]
[in a new window]
  		Figure 1. Interleukin (IL)-6 production during cholestasis. IL-6 and ß-actin messenger RNA levels (A) in liver and mean (± SEM) levels of IL-6 protein in plasma (B) and liver (C) of IL-6+/+ mice after bile duct ligation (BDL) or sham operation (n = 8 per group at each time point). Patterned bars, IL-6+/+ mice after sham operation; solid bars, IL-6+/+ mice after BDL.

 
Neither sham surgery nor BDL was associated with mortality during a 1-month follow-up. BDL mice showed biochemical evidence of hepatocellular injury as reflected by elevated plasma levels of ALT and AST, and significant cholestasis as reflected by elevated plasma levels of bilirubin, alkaline phosphatase, and {gamma}-glutamyltransferase 2 weeks after surgery (Figure 2) . However, significant differences between IL-6+/+ and IL-6-/- mice were not found. Sham operation did not affect normal liver parenchyma of IL-6+/+ and IL-6-/- mice up to 14 days after surgery as determined by histopathological examination. In contrast, BDL led to typical cholestatic changes in liver parenchyma that were similar in both mouse strains (Figure 3) .



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. IL-6 is not involved in cholestatic liver injury after bile duct ligation. Mean ± SE levels of bilirubin (A), alkaline phosphatase (Alk. phosph.; B), {gamma}-glutamyltransferase ({gamma}-GT; C), alanine aminotransferase (ALT; D), and aspartate aminotransferase (AST; E) in plasma of IL-6+/+ mice (solid bars) and IL-6-/- mice (open bars) 2 weeks after BDL or sham operation (n = 8 per group). p Values indicate differences between BDL and sham-operated mice (either IL-6+/+ or IL-6-/-). Differences between IL-6+/+ and IL-6-/- mice within BDL and sham-operated groups were not significant.

 


View larger version (87K):
[in this window]
[in a new window]
  		Figure 3. Comparable histology in cholestatic IL-6+/+ and IL-6-/- mice. Representative liver sections from sham and BDL animals (n = 8) 14 days after operation (hematoxylin and eosin). Note the normal liver architecture in sham mice (A and B) compared with BDL mice (C and D). The latter exhibit infrequent focal areas of necrosis (arrowheads) and scarce cellular infiltrates with marked bile duct proliferation (arrows), comparable in both IL-6+/+ (A and B) and IL-6-/- mice (C and D). CV = central vein; PV = portal vein. Scale bar: 100 µm.

 
These data demonstrate that IL-6 is produced in the liver after BDL, but does not play an important role in cholestatic liver injury.

Increased Endotoxin-induced Lethality in BDL IL-6-/- Mice
Two weeks after BDL or sham treatment, mice were challenged with endotoxin and monitored for 24 hours. All mice that survived the endotoxin challenge during the first 24 hours proved to be permanent survivors. As shown in Figure 4 , during the first 6 hours after endotoxin challenge, death was not observed in any group. Moreover, endotoxin challenge in sham-operated mice was not associated with mortality at all at the endotoxin dose given. However, after BDL, both mouse strains demonstrated significant mortality after administration of endotoxin (both p < 0.01 versus sham). Importantly, a significant difference was found in mortality between both cholestatic mouse strains, that is, mortality was 81% (13 of 16) in IL-6-/- mice and only 44% (7 of 16) in IL-6+/+ mice (p < 0.05).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 4. Increased lethality in endotoxin-treated BDL IL-6-/- mice. Endotoxin-induced lethality in BDL and sham IL-6+/+ and IL-6-/- mice (n = 16 per group). Two weeks after operation, mice were injected with endotoxin (4 µg/kg body weight, intraperitoneal), and survival was assessed every hour for 24 hours. Solid squares, IL-6+/+ mice, sham operated; open squares, IL-6-/- mice, sham operated; solid circles, IL-6+/+ mice, BDL; open circles, IL-6-/- mice, BDL.

 
Increased Cytokine Release in BDL IL-6-/- Mice after Endotoxin Challenge
After endotoxin challenge, IL-1{alpha}, IL-1ß, IL-6, and TNF-{alpha} levels were measured in plasma at various time intervals during the first 6 hours, that is, before any animal died. Proinflammatory cytokine levels were significantly higher in BDL than in sham mice (all p values < 0.05; Figure 5) . As compared with endotoxemic sham mice, IL-1{alpha} levels were 10-fold higher, IL-1ß levels were up to 30-fold higher, IL-6 levels were 10-fold higher, and TNF-{alpha} levels were 6-fold higher in endotoxemic cholestatic mice.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 5. Enhanced cytokine release in endotoxemic IL-6-/- mice. Mean (± SE) plasma cytokine levels after injection of endotoxin in IL-6+/+ and IL-6-/- mice (n = 8 per group) 2 weeks after sham or BDL treatment; plasma samples were obtained at the time points indicated. IL-1{alpha} (A and B), IL-1ß (C and D), IL-6 (E and F), and tumor necrosis factor-{alpha} (TNF; G and H) concentrations were determined by ELISA. IL-6 was not detectable in IL-6-/- mice (E and F). Note the differences in scale of the y axes for sham and BDL mice. Solid squares, IL-6+/+ mice, sham operated; open squares, IL-6-/- mice, sham operated; solid circles, IL-6+/+ mice, BDL; open circles, IL-6-/- mice, BDL. *p < 0.05 versus BDL IL-6+/+ mice.

 
Overall, IL-6-/- mice demonstrated a relatively higher release of cytokines on exposure to endotoxin as compared with IL-6+/+ mice. The difference in cytokine release between the two mouse strains was especially apparent in BDL mice.

Increased Endotoxin-induced Liver Damage in BDL IL-6-/- Mice
Endotoxin challenge resulted in influx of neutrophils in the liver, development of hepatocellular necrosis, increased plasma ALT levels, and increased liver edema (Figures 6 and 7) , which were more pronounced in BDL mice as compared with sham mice (p < 0.05 for all responses). After BDL and subsequent challenge with endotoxin, neutrophil influx, hepatocellular necrosis, rises in plasma ALT levels, and increased liver edema were all significantly more pronounced in IL-6-/- mice than in IL-6+/+ mice (p < 0.05 for all responses), whereas in sham animals these responses were not significantly different between IL-6+/+ and IL-6-/- mice, except for the higher liver wet-to-dry ratio in IL-6-/- mice (p < 0.05). Bilirubin levels 6 hours after endotoxin challenge did not increase above the high levels already present as a consequence of BDL in either mouse strain (data not shown).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 6. Increased endotoxin-induced liver damage in BDL IL-6-/- mice. Mean (± SE) neutrophil influx detected by chloroacetate esterase activity (A) and area of necrosis (B) in liver, plasma level of ALT (C), and liver edema (D) in IL-6+/+ mice (solid bars) and IL-6-/- mice (open bars) (n = 8 per group) 2 weeks after sham or BDL and 6 hours after endotoxin challenge. HPF = high-power field; PMNs = polymorphonuclear cells. *p < 0.05, BDL versus sham mice; {dagger} p < 0.05, IL-6-/- versus IL-6+/+, BDL mice; {ddagger} p < 0.05, IL-6-/- versus IL-6+/+, sham mice.

 


View larger version (96K):
[in this window]
[in a new window]
  		Figure 7. Increased endotoxin-induced liver pathology in BDL IL-6-/- mice. Representative liver sections from sham (A and B) and BDL (C and D) animals (n = 8 per group) 6 hours after administration of endotoxin (H&E; original magnification, x200). Necrosis was quantitated by measuring pixel intensities. Note the relatively normal liver architecture in sham-operated mice as compared with BDL mice, with infrequent focal areas of necrosis (arrowheads) and scarce cellular infiltrates (arrows); most necrosis and cellular infiltrates are found in IL-6-/- mice after BDL (C) and less necrosis and infiltrates are found in IL-6+/+ mice after BDL (D). CV = central vein; PV = portal vein.

 
Increased Endotoxin-induced Lung Inflammation in BDL IL-6–deficient Mice
Six hours after endotoxin challenge, IL-6 levels in the lungs of cholestatic IL-6+/+ mice were significantly elevated as compared with sham-operated IL-6+/+ mice (p < 0.05; Figure 8A) . IL-6 was not detectable in IL-6-/- mice. Endotoxin challenge resulted in influx of neutrophils in the lungs, an increase in myeloperoxidase and TNF-{alpha} levels in homogenized lung tissue, and increased lung edema (Figures 8B–8E), which were more pronounced in BDL mice than in sham-operated mice (p < 0.01 for all responses). After challenge with endotoxin, neutrophil influx, myeloperoxidase levels in lung tissue, and lung edema were all significantly more pronounced in IL-6-/- mice than in IL-6+/+ mice (p < 0.05 for all responses), whereas TNF-{alpha} levels were significantly higher only in BDL IL-6-/- mice (p < 0.05). Lung pathology was exaggerated in cholestatic mice as compared with sham-operated mice (p < 0.05). However, no difference was found between cholestatic IL-6+/+ and IL-6-/- mice (Figures 8F and 9) .



View larger version (39K):
[in this window]
[in a new window]
  		Figure 8. Increased endotoxin-induced lung damage in BDL IL-6-/- mice. Mean (± SE) plasma level of IL-6 in IL-6+/+ mice (A; IL-6 was not detectable in IL-6-/- mice), mean (± SE) neutrophil count in BALF (B), myeloperoxidase (MPO) levels in lungs (C), lung edema (D), lung TNF-{alpha} levels (E), and lung histology score (F) in IL-6+/+ mice (solid columns) and IL-6-/- mice (open columns) (n = 8 per group) 2 weeks after sham or BDL treatment and 6 hours after endotoxin challenge. *p < 0.01, BDL versus sham mice; {dagger} p < 0.05, IL-6-/- versus IL-6+/+, BDL mice; {ddagger} p < 0.05, IL-6-/- versus IL-6+/+, sham mice.

 


View larger version (99K):
[in this window]
[in a new window]
  		Figure 9. Increased endotoxin-induced pathology in lungs of BDL mice compared with sham mice. Representative sections of lungs with anti-granulocyte immunostaining of IL-6+/+ and IL-6-/- mice (n = 8) 2 weeks after BDL or sham surgery and 6 hours after endotoxin challenge. BDL mice displayed more intense and diffuse inflammatory infiltrates, with more intense staining for PMNs (arrows) and thrombotic areas (arrowheads); the absence of IL-6, however, did not affect lung pathology in sham or BDL mice. Scale bar: 100 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Obstructive jaundice is associated with enhanced susceptibility to toxic effects of endotoxin (the present study, and References 11, 20, and 25–28). Increased endotoxin responsiveness has been implicated in the pathogenesis of postoperative complications that frequently occur in patients with extrahepatic biliary obstruction, including sepsis and the systemic inflammatory response syndrome (5, 6, 29). In the present study, we determined the role of endogenous IL-6 in the development of hepatic inflammation and injury after biliary obstruction, and the increased susceptibility to endotoxin-induced toxicity associated with obstructive jaundice in a mouse model. The main findings were that IL-6 does not play an important role in hepatic pathology resulting from biliary obstruction during a 2-week follow-up, but that this cytokine contributes to a significant extent to the occurrence of hepatocellular damage, lung inflammation, and death after endotoxin challenge in jaundiced mice. These data suggest that endogenous IL-6 plays an important role in the increased endotoxin responsiveness during cholestasis.

Our results were obtained with IL-6-/- mice on a BALB/c background originating from the Basel Institute for Immunology, and IL-6+/+ BALB/c mice purchased from a commercial supplier. Thus, although unlikely, we cannot exclude that genetic drift has occurred between these two groups of mice so that other genes could have impacted on outcome parameters.

IL-6 is a cytokine with many different functional properties that can be produced by a variety of cell types. Patients with obstructive jaundice have elevated IL-6 levels in their circulation, which decrease after cholangio-drainage (30, 31). In accordance, IL-6 levels were increased in plasma and liver homogenates during BDL-induced jaundice in the present study, confirming previous investigations (11, 15, 16, 32, 33). This endogenously produced IL-6 did not contribute to cholestatic liver damage ensuing from extrahepatic obstruction. Indeed, each parameter measured 2 weeks after BDL, that is, transaminases, alkaline phosphatase, {gamma}-glutamyltransferase in plasma, and histopathology of the liver, proved to be similar in IL-6-/- and IL-6+/+ mice. Our findings are in line with a study in which IL-6-/- mice did not demonstrate clear phenotypic differences 1 week after BDL when compared with IL-6+/+ mice (16). However, IL-6 may play a role in more chronic cholestatic liver injury, as was indicated by observations that IL-6-/- mice developed a more advanced stage of biliary fibrosis 12 weeks after BDL, which was associated with higher serum bilirubin levels and mortality (32).

Extrahepatic cholestasis resulted in a markedly increased susceptibility to endotoxin, as indicated by mortality after administration of an endotoxin dose that did not cause mortality in sham-operated mice, enhanced cytokine release, and more severe liver damage and lung inflammation. These data confirm and extend data of previous studies on BDL rodents (3, 28, 33, 34). Although the hepatobiliary alterations in BDL IL-6-/- mice were indistinguishable from those in BDL IL-6+/+ mice, the susceptibility of the former mice to systemic endotoxin exposure was more enhanced as compared with BDL wild-type mice. The role of IL-6 in endotoxin-induced inflammation has been investigated in a number of studies with nonjaundiced IL-6-/- mice. Similar or slightly increased mortality rates and enhanced release of TNF after systemic endotoxin administration have been reported, when compared with IL-6+/+ mice (35, 36). In line with these data, we found in the present study that overt differences between IL-6-/- and IL-6+/+ mice did not occur after endotoxin challenge and sham surgery, although IL-6 deficiency was associated with marginally elevated circulating levels of TNF and IL-1. The modest antiinflammatory role of IL-6 during endotoxemia is explained at least in part by the fact that this cytokine is capable of inhibiting endotoxin-induced TNF and IL-1 production by mononuclear cells in vitro, and reducing TNF release in endotoxemic mice in vivo (37, 38). Remarkably, the role of IL-6 in endotoxin-induced tissue damage and mortality was much more important in cholestatic mice, that is, IL-6-/- mice injected with endotoxin displayed more extensive liver injury and lung inflammation, and higher mortality rates than did BDL IL-6+/+ mice 2 weeks after BDL. The sequestration of neutrophils in the pulmonary microvasculature proved to be an important initiator of acute lung inflammation as described previously (39). Thus, obstructive jaundice results in an endotoxin hypersensitivity state in which endogenous IL-6 activity has become an important protector of endotoxin-induced damage. Interestingly, we did a similar observation on the role of endogenous IL-1, albeit in the opposite direction. Indeed, IL-1 receptor–deficient mice, which lack the capacity to transmit signals of both IL-1{alpha} and IL-1ß, were equally sensitive to endotoxin as wild-type mice in the absence of jaundice, but were partially protected against the toxic effects of endotoxin after BDL, as reflected by attenuated cytokine release, less severe liver injury, and reduced mortality (11). Taken together, these data indicate that IL-1{alpha}, IL-1ß, and IL-6, produced in the liver after BDL, are part of a delicate balance between proinflammatory and antiinflammatory mediators, the former of which (including IL-1{alpha} and IL-1ß) mediate toxicity provoked by endotoxin, whereas the latter (including IL-6) play a protective role. These two sides of the cytokine network also influence each other's activity during cholestasis and endotoxemia, as indicated by our findings that cholestatic IL-1 receptor–deficient mice displayed lower IL-6 levels after endotoxin challenge (11), whereas cholestatic IL-6-/- mice demonstrated higher IL-1{alpha} and IL-1ß levels during endotoxemia.

Cholestatic IL-6-/- mice showed more extensive lung inflammation than IL-6+/+ mice did after intraperitoneal administration of endotoxin. During cholestasis a local decrease in hepatic blood flow predisposes to lung inflammation by augmenting circulating levels of endotoxin and tumor necrosis factor (40). In a previous study, IL-6-/- mice without jaundice demonstrated enhanced influx of neutrophilic granulocytes into BALF and lung tissue after administration of endotoxin per aerosol than IL-6+/+ mice (36). We found a similar antiinflammatory role of endogenous IL-6 in the pulmonary compartment after intranasal administration of lipoteichoic acid, a major component of the gram-positive bacterial cell wall, as reflected by enhanced inflammation in lungs of IL-6-/- mice (18). Notably, the role of IL-6 in lung inflammation may vary depending on the inflammatory stimulus used, for example, inflammatory responses induced by peptidoglycans were diminished in lungs of IL-6-/- mice (18). Although a clear explanation for the differential role of IL-6 in various types of lung inflammation is lacking, it is clear that endotoxin and peptidoglycan stimulate the innate immune system via different pathways. Indeed, while endotoxin triggers Toll-like receptor (TLR)-4, peptidoglycan initiates an inflammatory response via TLR-2. In more complex situations, such as during pneumonia or lung injury induced by sepsis or systemic endotoxin exposure, multiple pattern recognition receptors may be stimulated in the lung, triggering different signaling pathways, and it is conceivable that the net role of IL-6 in such complex responses may vary from condition to condition (41–43). In addition, it should be noted that IL-6 deficiency did not influence lung histology scores, although IL-6-/- mice did display elevated lung TNF-{alpha} levels, and increased pulmonary neutrophil influx and edema. Possibly, differences in lung histology may have occurred later than after 6 hours (the time of sacrifice) (44), and thus our design may not have been appropriate to demonstrate differences between IL-6-/- and IL-6+/+ mice in this respect. However, histologic examination of lungs at time points beyond 6 hours was not feasible, considering that in particular IL-6-/- mice rapidly died after this time period, presumably as a result of systemic toxicity rather than lung injury.

In conclusion, we demonstrated here that IL-6, produced in the liver during obstructive jaundice induced by BDL, does not contribute to hepatic inflammation and injury during a 2-week follow-up. However, enhanced IL-6 release is found in jaundiced mice exposed to endotoxin, and eliminating this IL-6 response renders mice hypersensitive to the proinflammatory and lethal effects of endotoxin. These results suggest that endogenous IL-6 plays an important role in protecting the cholestatic host against hypersensitivity to endotoxin.


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

Conflict of Interest Statement: M.E.S. has no declared conflict of interest; T.V.D.P. has no declared conflict of interest; C.J.F.V.N. has no declared conflict of interest; F.J.W.T.K. has no declared conflict of interest; D.J.G. has no declared conflict of interest.

Received in original form March 3, 2003; accepted in final form November 3, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sewnath ME, Birjmohun RS, Rauws EA, Huibregtse K, Obertop H, Gouma DJ. The effect of preoperative biliary drainage on postoperative complications after pancreaticoduodenectomy. J Am Coll Surg 2001;192:726–734.[CrossRef][Medline]
  2. Sewnath ME, Karsten TM, Prins MH, Rauws EJ, Obertop H, Gouma DJ. A meta-analysis on the efficacy of preoperative biliary drainage for tumors causing obstructive jaundice. Ann Surg 2002;236:17–27.[CrossRef][Medline]
  3. Chang SW, Ohara N. Pulmonary circulatory dysfunction in rats with biliary cirrhosis. An animal model of the hepatopulmonary syndrome. Am Rev Respir Dis 1992;145:798–805.[Medline]
  4. Kimmings AN, van Deventer SJ, Obertop H, Rauws EA, Gouma DJ. Inflammatory and immunologic effects of obstructive jaundice: pathogenesis and treatment. J Am Coll Surg 1995;181:567–581.[Medline]
  5. Ingoldby CJ, McPherson GA, Blumgart LH. Endotoxemia in human obstructive jaundice: effect of polymyxin B. Am J Surg 1984;147:766–771.[CrossRef][Medline]
  6. Hunt DR, Allison ME, Prentice CR, Blumgart LH. Endotoxemia, disturbance of coagulation, and obstructive jaundice. Am J Surg 1982;144:325–329.[CrossRef][Medline]
  7. Pain JA, Bailey ME. Measurement of operative plasma endotoxin levels in jaundiced and non-jaundiced patients. Eur Surg Res 1987;19:207–216.[Medline]
  8. Cahill CJ. Prevention of postoperative renal failure in patients with obstructive jaundice: the role of bile salts. Br J Surg 1983;70:590–595.[Medline]
  9. Pitt HA, Cameron JL, Postier RG, Gadacz TR. Factors affecting mortality in biliary tract surgery. Am J Surg 1981;141:66–72.[CrossRef][Medline]
  10. Greve JW, Gouma DJ, Soeters PB, Buurman WA. Suppression of cellular immunity in obstructive jaundice is caused by endotoxins: a study with germ-free rats. Gastroenterology 1990;98:478–485.[Medline]
  11. Sewnath ME, van der Poll T, ten Kate JWF, Van Noorden CJ, Gouma DJ. Interleukin-1 receptor type I gene–deficient bile duct-ligated mice are partially protected against endotoxin. Hepatology 2002;35:149–158.[CrossRef][Medline]
  12. van der Poll T, van Deventer SJ. The role of interleukin 6 in endotoxin-induced inflammatory responses. Prog Clin Biol Res 1998;397:365–377.[Medline]
  13. Streetz KL, Luedde T, Manns MP, Trautwein C. Interleukin 6 and liver regeneration. Gut 2000;47:309–312.[Free Full Text]
  14. Gauldie J, Richards C, Harnish D, Lansdorp P, Baumann H. Interferon ß2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 1987;84:7251–7255.[Abstract/Free Full Text]
  15. Bemelmans MH, Gouma DJ, Greve JW, Buurman WA. Cytokines tumor necrosis factor and interleukin-6 in experimental biliary obstruction in mice. Hepatology 1992;15:1132–1136.[Medline]
  16. Liu Z, Sakamoto T, Yokomuro S, Ezure T, Subbotin V, Murase N, Contrucci S, Demetris AJ. Acute obstructive cholangiopathy in interleukin-6 deficient mice: compensation by leukemia inhibitory factor (LIF) suggests importance of gp-130 signaling in the ductular reaction. Liver 2000;20:114–124.[CrossRef][Medline]
  17. Sewnath ME, van der Poll T, Van Noorden CJ, ten Kate FJ, Gouma DJ. Endogenous interferon {gamma} protects against cholestatic liver injury in mice. Hepatology 2002;36:1466–1477.[CrossRef][Medline]
  18. Leemans JC, Vervoordeldonk MJ, Florquin S, van Kessel KP, van der Poll T. Differential role of interleukin-6 in lung inflammation induced by lipoteichoic acid and peptidoglycan from Staphylococcus aureus. Am J Respir Crit Care Med 2002;165:1445–1450.[Abstract/Free Full Text]
  19. Bauer J, Ganter U, Geiger T, Jacobshagen U, Hirano T, Matsuda T, Kishimoto T, Andus T, Acs G, Gerok W. Regulation of interleukin-6 expression in cultured human blood monocytes and monocyte-derived macrophages. Blood 1988;72:1134–1140.[Abstract/Free Full Text]
  20. Sewnath ME, Levels JHM, Oude Elferink RP, Van Noorden CJF, ten Kate JWF, van Deventer SJH, Gouma DJ. Endotoxin-induced mortality in bile duct-ligated rats after administration of reconstituted high-density lipoprotein. Hepatology 2000;32:1289–1299.[CrossRef][Medline]
  21. Ferrer TJ, Webb JW, Wallace BH, Bridges CD, Palmer HE, Robertson RD, Cone JB. Interleukin-10 reduces morbidity and mortality in murine multiple organ dysfunction syndrome (MODS). J Surg Res 1998;77:157–164.[CrossRef][Medline]
  22. Olszyna DP, Florquin S, Sewnath M, Branger J, Speelman P, van Deventer SJ, Strieter RM, van der Poll T. CXC chemokine receptor 2 contributes to host defense in murine urinary tract infection. J Infect Dis 2001;184:301–307.[CrossRef][Medline]
  23. van der Poll T, Marchant A, Buurman WA, Berman L, Keogh CV, Lazarus DD, Nguyen L, Goldman M, Moldawer LL, Lowry SF. Endogenous IL-10 protects mice from death during septic peritonitis. J Immunol 1995;155:5397–5401.[Abstract]
  24. Hadjiminas DJ, McMasters KM, Robertson SE, Cheadle WG. Enhanced survival from cecal ligation and puncture with pentoxifylline is associated with altered neutrophil trafficking and reduced interleukin-1ß expression but not inhibition of tumor necrosis factor synthesis. Surgery 1994;116:348–355.[Medline]
  25. Kimmings AN, van Deventer SJ, Obertop H, Gouma DJ. Treatment with recombinant bactericidal/permeability-increasing protein to prevent endotoxin-induced mortality in bile duct-ligated rats. J Am Coll Surg 1999;189:374–379.[CrossRef][Medline]
  26. Kimmings N, Sewnath ME, Mairuhu WM, van Zanten AP, Rauws EA, van Deventer SJ, Gouma DJ. The abnormal lipid spectrum in malignant obstructive jaundice in relation to endotoxin sensitivity and the result of preoperative biliary drainage. Surgery 2001;129:282–291.[CrossRef][Medline]
  27. Greve JW, Gouma DJ, Buurman WA. Complications in obstructive jaundice: role of endotoxins. Scand J Gastroenterol Suppl 1992;194:8–12.[Medline]
  28. Chang SW, Ohara N. Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 1994;94:2009–2019.
  29. Kimmings AN, van Deventer SJH, Obertop H, Rauws EAJ, Huibregtse K, Gouma DJ. Endotoxin, cytokines, and endotoxin binding proteins in obstructive jaundice and after preoperative biliary drainage. Gut 2000;46:725–731.[Abstract/Free Full Text]
  30. Akiyama T, Hasegawa T, Sejima T, Sahara H, Seto K, Saito H, Takashima S. Serum and bile interleukin 6 after percutaneous transhepatic cholangio-drainage. Hepatogastroenterology 1998;45:665–671.[Medline]
  31. Kimura F, Miyazaki M, Suwa T, Sugiura T, Shinoda T, Itoh H, Ambiru S, Shimizu H, Nakagawa K. Serum interleukin-6 levels in patients with biliary obstruction. Hepatogastroenterology 1999;46:1613–1617.[Medline]
  32. Ezure T, Sakamoto T, Tsuji H, Lunz JG, Murase N, Fung JJ, Demetris AJ. The development and compensation of biliary cirrhosis in interleukin-6–deficient mice. Am J Pathol 2000;156:1627–1639.[Abstract/Free Full Text]
  33. Lechner AJ, Velasquez A, Knudsen KR, Johanns CA Jr, Tracy TF, Matuschak GM. Cholestatic liver injury increases circulating TNF-{alpha} and IL-6 and mortality after Escherichia coli endotoxemia. Am J Respir Crit Care Med 1998;157:1550–1558.
  34. O'Neil S, Hunt J, Filkins J, Gamelli R. Obstructive jaundice in rats results in exaggerated hepatic production of tumor necrosis factor-{alpha} and systemic and tissue tumor necrosis factor-{alpha} levels after endotoxin. Surgery 1997;122:281–286.[CrossRef][Medline]
  35. Dalrymple SA, Slattery R, Aud DM, Krishna M, Lucian LA, Murray R. Interleukin-6 is required for a protective immune response to systemic Escherichia coli infection. Infect Immun 1996;64:3231–3235.[Abstract]
  36. Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei XF, Achong MK. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest 1998;101:311–320.[Medline]
  37. Aderka D, Le JM, Vilcek J. IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol 1989;143:3517–3523.[Abstract]
  38. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA. Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 1990;75:40–47.[Abstract/Free Full Text]
  39. Suwa T, Hogg JC, Klut ME, Hards J, van Eeden SF. Interleukin-6 changes deformability of neutrophils and induces their sequestration in the lung. Am J Respir Crit Care Med 2001;163:970–976.[Abstract/Free Full Text]
  40. 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.[Abstract/Free Full Text]
  41. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–376.[CrossRef][Medline]
  42. Maus U, Huwe J, Maus R, Seeger W, Lohmeyer J. Alveolar JE/MCP-1 and endotoxin synergize to provoke lung cytokine upregulation, sequential neutrophil and monocyte influx, and vascular leakage in mice. Am J Respir Crit Care Med 2001;164:406–411.[Abstract/Free Full Text]
  43. Leemans JC, Heikens M, van Kessel KP, Florquin S, van der Poll T. Lipoteichoic acid and peptidoglycan from Staphylococcus aureus synergistically induce neutrophil influx into the lungs of mice. Clin Diagn Lab Immunol 2003;10:950–953.[Abstract/Free Full Text]
  44. Davidson KG, Bersten AD, Barr HA, Dowling KD, Nicholas TE, Doyle IR. Endotoxin induces respiratory failure and increases surfactant turnover and respiration independent of alveolocapillary injury in rats. Am J Respir Crit Care Med 2002;165:1516–1525.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
D. Angus, A. Ishizaka, M. Matthay, F. Lemaire, W. MacNee, and E. Abraham
Critical Care in AJRCCM 2004
Am. J. Respir. Crit. Care Med., March 15, 2005; 171(6): 537 - 544.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200303-311OCv1
169/3/413    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sewnath, M. E.
Right arrow Articles by Gouma, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sewnath, M. E.
Right arrow Articles by Gouma, D. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2004 American Thoracic Society