Ischemic preconditioning affects interleukin release in fatty livers of rats undergoing ischemia/reperfusion

Authors

  • Anna Serafín,

    1. Experimental Pathology Department, Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Cientificas, Institut d′Investigacions Biomediques August Pi i Sunyer, Barcelona
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  • Joan Roselló-Catafau,

    Corresponding author
    1. Experimental Pathology Department, Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Cientificas, Institut d′Investigacions Biomediques August Pi i Sunyer, Barcelona
    • Department of Experimental Pathology, Institut d′Investigacions Biomediques August Pi i Sunyer, C/Rosello161,7a planta, 08036-Barcelona, Spain
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    • fax: +34- 933638301

  • Neus Prats,

    1. Veterinary School, Universitat Autonoma de Barcelona, Barcelona
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  • Emilio Gelpí,

    1. Experimental Pathology Department, Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Cientificas, Institut d′Investigacions Biomediques August Pi i Sunyer, Barcelona
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  • Joan Rodés,

    1. Liver Unit, Hospital Clínic, Universitat de Barcelona, Barcelona, Spain
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  • Carmen Peralta

    1. Experimental Pathology Department, Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Cientificas, Institut d′Investigacions Biomediques August Pi i Sunyer, Barcelona
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Abstract

The present study evaluates the effect of ischemic preconditioning on interleukin-1 (IL-1) and interleukin-10 (IL-10) generation following hepatic ischemia/reperfusion (I/R) in normal and steatotic livers as well as the role of nitric oxide (NO) in this process. Increased IL-1β and IL-10 levels were observed in normal livers after I/R. Steatotic livers showed higher IL-1β levels than normal livers, and IL-10 at control levels. The injurious role of IL-1β and the benefits of IL-10 on hepatic I/R injury was shown with the use of IL-1 receptor antagonist (IL-1ra), anti-IL-10 polyclonal antibody against IL-10 (anti-IL-10) and exogenous IL-10. The effective dose of these treatments was different in both types of livers. Preconditioning prevented IL-1β release and increased IL-10 generation after I/R in normal and steatotic livers. IL-1β or anti-IL-10 pretreatments reversed the benefits of preconditioning. IL-1β action inhibition in a preconditioned group that was pretreated with anti-IL-10 did not modify the benefits of preconditioning. In addition, anti-IL-10 pretreatment in the preconditioned group resulted in IL-1β levels comparable to those observed after I/R. NO inhibition eliminated the benefits of preconditioning on IL-10 release, IL-1β levels, and hepatic injury. In conclusion, preconditioning, through IL-10 overproduction, inhibits IL-1β release and the ensuing hepatic I/R injury in normal and steatotic livers. IL-10 generation induced by preconditioning could be mediated by NO. (HEPATOLOGY 2004;39:688–698.)

The presence of fatty metamorphosis reduces the tolerance of the liver to ischemia/reperfusion (I/R) injury. Liver resection in patients with steatosis is associated with a risk of postoperative mortality when compared to subjects with nonfatty livers.1, 2 In addition, the use of steatotic livers for transplantation is associated with an increased risk of primary nonfunction or dysfunction after surgery.3, 4

Interleukin-1 (IL-1) is primarily involved in promoting inflammatory processes, and blockade of IL-1 action reduces tissue injury and mortality after hepatic I/R in nonsteatotic livers.5–8 In contrast, interleukin-10 (IL-10) show protective effects in nonsteatotic livers undergoing warm ischemia, and treatment with IL-10 increased allograft survival in liver transplantation.9–12 Lipopolysaccharide (LPS) administration leads to similar IL-1 levels in both normal and obese mice, whereas lower levels of IL-10 and IL-1 receptor antagonist (IL-1ra) were detected after LPS induction in obese as compared to normal mice.13 LPS induction of hepatic IL-10 messenger RNA expression was also abnormal in obese rats.14 Considering the key role of ILs in hepatic I/R injury in nonsteatotic livers and the differences in IL levels found between obese and normal animals after LPS induction, an altered equilibrium of pro- and anti-inflammatory mediators could be responsible for the increased vulnerability of steatotic liver to hepatic I/R injury. Thus, high IL-1 levels and/or a reduced ability to generate IL-10 in obese animals may be associated with the development of more severe hepatic injury. This possibility was considered in the present study.

A recent article reported that ischemic preconditioning, where brief periods of ischemia and reperfusion before a prolonged ischemic period, protects steatotic livers during normothermic conditions.15 Although the mechanisms by which preconditioning provides protection remain unknown, organ protection depends on the release of endothelial substances such as nitric oxide (NO).15 The existence of a relationship between NO generation and IL production in normal livers has been previously demonstrated in different inflammatory processes.16–18 Thus, the inhibition of NO synthesis increased IL-1 generation following hepatic I/R,16, 17 and inducible nitric oxide synthase (iNOS)-deficient animals exhibited exaggerated plasma IL-1 levels in response to LPS.18 In addition, the induction of IL-10 production by NO has been reported in myocardial ischemia.19 The possibility that NO, implicated in preconditioning, could modulate the increased vulnerability of fatty livers to hepatic I/R through its action on IL release remains to be elucidated.

In this article, we report the results of an experimental study whose aims were to evaluate (1) the role of both IL-1 and IL-10 in hepatic I/R in normal and fatty livers, and (2) the effect of ischemic preconditioning on IL-1 and IL-10 generation following hepatic I/R in both types of livers as well as the role of NO in this process.

Abbreviations:

I/R, ischemia/reperfusion; IL, interleukin; LPS, lipopolysaccharide; IL-1ra, IL-1 receptor antagonist; NO, nitric oxide; iNOS, inducible nitric oxide synthase; Ob, obese; Ln, lean; PC, preconditioning; NAME, N-nitro-L-arginine methylester; AG, aminoguanidine hemisulfate; anti-IL-10, anti-IL-10 polyclonal antibody; MDA, malondialdehyde; ALT, alanine transaminase; ROS, reactive oxygen species; cNOS, constitutive nitric oxide synthase; TNF, tumor necrosis factor-α; NF-κB, nuclear factor-κB; AP-1, activator protein 1; p38 MAPK, p38 mitogen1-activated protein kinase.

Materials and Methods

Experimental Animals.

Homozygous (obese, Ob) and heterozygous (lean, Ln) Zucker rats, 16 to 18 weeks of age (Iffa-Credo, L′Abresle, France), were used for the experiments. Ob Zucker rats showed severe macrovesicular and microvesicular fatty infiltration in hepatocytes (between 60 % and 70% steatosis). In contrast, Ln Zucker rats showed no evidence of steatosis (Fig. 1).

Figure 1.

Difference of steatosis in the livers of Ob vs. Ln Zucker rats using red-oil staining. (A) Ln Zucker rats show no evidence of steatosis. (B) Ob Zucker rats show fatty infiltration in hepatocytes. Original magnification x270.

A model of segmental (70%) hepatic ischemia was used. Rats were anesthetized with ketamine (100 mg/kg) and xylazine (8 mg/kg). After a midline laparotomy, the hepatic artery and portal vein to the left and the median liver lobes were occluded with an atraumatic vascular clamp for the period of ischemia under study. Reperfusion was initiated by removal of the clamp.15, 20 Experiments to assess animal survival were performed by resecting the nonischemic lobes (30%) at the time of reperfusion.15, 21

This study was performed in concordance with European Union regulations (Directive 86/609 EEC) for animal experiments.

Experimental Design

Protocol I.

To evaluate the effect of preconditioning on IL release after hepatic reperfusion, the following experimental groups were set up:

  • 1Control (C; n = 12): Ln and Ob animals (6 in each group) were subjected to anesthesia and laparotomy.
  • 2I/R (n = 12): Ln and Ob animals (6 in each group) were subjected to 60 minutes of partial ischemia followed by 6 hours of reperfusion.
  • 3Preconditioning + I/R (PC + I/R;n = 12): Same as group 2 but with previous preconditioning induced by 5 minutes of ischemia followed by 10 minutes of reperfusion.
  • 4PC + I/R + NAME (n = 12): Same as group 3 but treated with N-nitro-L-arginine methylester (NAME), a NO-synthesis inhibitor (Sigma Chemical Co., St. Louis, MO), at doses of 10 mg/kg (i.v.) 5 minutes before preconditioning.15, 22
  • 5PC + I/R + AG (n = 12): Same as group 3 but treated with aminoguanidine hemisulfate (AG), an inducible nitric oxide synthase inhibitor (iNOS) (Sigma Chemical, St. Louis, MO), at doses of 10 mg/kg, i.v., 5 minutes before preconditioning.23, 24
  • 6I/R + IL-1ra (n = 24): Same as group 2 but treated with IL-1ra (Amgen Biologicals, Thousand Oaks, CA) at doses (i.v.) of 40 mg/kg (n = 12; 6 Ln and 6 Ob) and 80 mg/kg (n = 12; 6 Ln and 6 Ob) 5 minutes before ischemia.5, 6
  • 7PC + I/R + IL-1β (n = 24): Same as group 3 but treated with recombinant rat IL-1β (Peprotech EC Ltd., Rocky Hill, NJ) at doses (i.v.) of 10 μg/kg (n = 12; 6 Ln and 6 Ob) and 20 μg/kg (n = 12; 6 Ln and 6 Ob) 5 minutes before preconditioning.25
  • 8I/R + anti-IL-10 (n = 6): Ln animals subjected to I/R (as in group 2) were treated with an anti-IL-10 polyclonal antibody (anti-IL-10) directed against rat IL-10 (Biosource International, Camarillo, CA), at doses of 0.5 mg/kg (i.v.) 5 minutes before ischemia.26
  • 9I/R + IL-10 (n = 36): Same as group 2 but treated with recombinant rat IL-10 (Peprotech EC Ltd.) at doses (i.v.) of 3 μg/kg (n = 12; 6 Ln and 6 Ob), 10 μg/kg (n = 12; 6 Ln and 6 Ob) and 30 μg/kg (n = 12; 6 Ln and 6 Ob), 5 minutes before ischemia.10, 11
  • 10PC + I/R + anti-IL-10 (n = 12): Same as group 3 but treated with anti-IL-10 at doses of 0.5 mg/kg (i.v.) 5 minutes before preconditioning.
  • 11PC + I/R + anti-IL-10 + IL-1ra (n = 12): Same as group 3 but treated with anti-IL-10 (0.5 mg/kg) and IL-1ra (40 mg/kg in Ln and 80 mg/kg in Ob) 5 minutes before preconditioning.

Protocol II.

To evaluate necrotic cell death, animals subjected to the same experimental procedures as in groups 2 to 11 (protocol I), were subjected to 24 hours of reperfusion after 60 minutes of ischemia (groups 12-21).

Blood and liver samples were obtained after reperfusion. Evaluation of hepatic injury was performed by determination of transaminases in plasma and histological analysis of the liver. Hepatic levels of IL-1α, IL-1β, and IL-10 were measured. Malondialdehyde (MDA), as an index of lipid peroxidation, was evaluated in liver tissue.

The entire experimental design (protocols I and II) is summarized in Fig. 2.

Figure 2.

Experimental protocol set up in both fatty and normal livers subjected to 60 minutes of partial ischemia (60m) followed by 6 hours or 24 hours of reperfusion (R).

Protocol III.

To establish the relevance of the protective effects of IL-10, survival experiments were carried out. Ln and Ob animals (10 in each group) were subjected to 60 minutes of total ischemia. Reperfusion was initiated by removal of the clamp, and survival was evaluated for 30 days after surgery. The effects of different treatments, including anti-IL10, exogenous IL-10, and preconditioning (with or without anti-IL-10), on survival in animals subjected to 60 minutes of total ischemia (10 Ln and 10 Ob in each group) was also evaluated. As in previous studies,15, 21 survival was considered permanent it the rats were alive 30 days after surgery.

Biochemical Determinations

Alanine Transaminase (ALT).

ALT was evaluated in plasma according to standard procedures.

Interleukin Assays.

IL determination (IL-1α, IL-1β, and IL-10) in liver tissue were measured by enzyme-linked immunosorbent assay technique as previously reported.6, 27–29 Commercial kits from Amersham Life Science (Amersham, UK) were used.

Lipid Peroxidation Assay.

Lipid peroxidation in liver samples was determined by the thiobarbiturate reaction measuring the formation of MDA.30

Histology.

For the severity of hepatic injury, hematoxylin and eosin-stained sections were evaluated by a point-counting method using an ordinal scale: grade 0 = minimal or no evidence of injury; grade 1 = mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis; grade 2 = moderate-to-severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, and loss of intercellular borders; and grade 3 = severe necrosis with disintegration of hepatic cords, hemorrhage, and neutrophil infiltration. Forty random sections were investigated per slide to determine the percentage of necrotic cells.15, 31 Steatosis in liver tissue was evaluated using red-oil staining on frozen specimens according to standard procedures.

Statistics.

Data are expressed as mean ± SD. Statistical comparison was performed with analysis of variance, followed by Student-Newman-Keuls tests. An associated probability of P < .05 was considered significant.

Results

IL-1α does not seem to play a role in mediating hepatic I/R injury because IL-1α levels after 6 hours of reperfusion were comparable to those obtained in the control group (Fig. 3A). In addition, peak levels of IL-1α were not found at earlier time points (data not shown). However, a significant increase in hepatic IL-1β levels was observed after I/R, and the values of IL-1β were approximately 2-fold higher in fatty than in normal livers (Fig. 3B). When preconditioning was performed (PC + I/R), IL-1β levels similar to those found in the control group were observed. With respect to IL-10, increased IL-10 levels were found in nonsteatotic livers following I/R, andcontrol levels of IL-10 were observed in fatty livers (Fig. 3C). Preconditioning resulted in significantly higher IL-10 levels in both types of livers than IL-10 levels observed in the I/R group.

Figure 3.

(A) IL-1α, (B) IL-1β, and (C) IL-10 levels in normal and steatotic livers at 6 hours of hepatic reperfusion. *P < .05 vs. Control. **P < .05 vs. I/R.

The involvement of IL-1β in the decreased tolerance of steatotic livers to I/R injury was evaluated. At 6 hours of reperfusion, the values of ALT were at least 4 times higher in fatty than in normal livers (Fig. 4A). In normal livers, the administration of IL-1 receptor antagonist (I/R + IL-1ra) at a dose of 40 mg/kg attenuated the increases in ALT levels observed after reperfusion; in fatty livers, this antagonist, at the same dose, was ineffective. In the presence of steatosis, a dose of 80 mg/kg was necessary to ameliorate the hepatic injury induced by I/R. Preconditioning was effective in both types of livers because ALT levels significantly lower than those obtained after I/R were found. IL-1β administration in the PC + I/R group (PC + I/R + IL-1β) at doses of 10 and 20 μg/kg in normal and steatotic livers, respectively, was able to completely reverse the benefits of preconditioning. An identical pattern was observed for the percentage of grade 3 necrosis (Fig. 4B and 4C). As shown in Fig. 4C, the percentage of necrosis at 24 hours in the unpreconditioned group was 18% in normal livers and 75% in steatotic livers. Preconditioning and IL-1 receptor antagonist pretreatment significantly reduced the percentage of necrosis found in the I/R group. The administration of IL-1β in the PC + I/R group abolished the benefits of preconditioning on grade 3 necrosis.

Figure 4.

Role of IL-1β in ischemic preconditioning. (A) ALT levels in Ln and Ob at 6 hours of reperfusion. (B and C) Percentage of grade 3 necrosis in Ln and Ob at 6 hours (B) and 24 hours (C) of reperfusion. *P < .05 vs. Control. **P < .05 vs. I/R. ***P < .05 vs. PC + I/R.

The role of IL-10 in hepatic I/R injury was evaluated. The protective role of endogenous IL-10 on I/R injury in normal livers was confirmed because the administration of anti-IL-10 polyclonal antibody (I/R + anti-IL-10) significantly increased the transaminase levels found after I/R (Fig. 5A). The exogenous IL-10 supplementation in the I/R group (I/R + IL-10) at a dose of 3 μg/kg did not modify the increases in ALT levels found in normal livers after hepatic I/R; however, a dose of 10 μg/kg reduced hepatic I/R injury. At a dose of 30 μg/kg, the ALT levels were similar to those observed after hepatic I/R. The ineffectiveness of a low dose of exogenous IL-10 and the adverse effects of a high dose of exogenous IL-10 have been reported previously in different experimental models of inflammation.10 In fatty livers, doses of 3 and 10 μg/kg did not modify the increases in ALT levels observed after I/R, but 30 μg/kg was able to confer protection against hepatic I/R injury (Fig. 5A). The administration of Abs against IL-10 in the PC + I/R group (PC + I/R + anti-IL-10) was able to completely reverse the benefits of preconditioning on the biochemical parameters of hepatic injury. However, IL-1β action inhibition in the PC + I/R + anti-IL-10 group (PC + I/R + anti-IL-10 + IL-1ra) resulted in transaminase levels similar to those observed when preconditioning was performed. A pattern similar to that for transaminase levels was observed for grade 3 necrosis (Fig. 5B and 5C).

Figure 5.

Role of IL-10 in ischemic preconditioning. (A) ALT levels in Ln and Ob at 6 hours of reperfusion. (B and C) Percentage of grade 3 necrosis in Ln and Ob at 6 hours (B) and 24 hours (C) of reperfusion. *P < .05 vs. Control. **P < .05 vs. I/R. ***P < .05 vs. PC + I/R.

Pretreatment with exogenous IL-10 (10 and 30 μg/kg in normal and steatotic livers, respectively) as preconditioning reduced IL-1β release following hepatic I/R (Fig. 6A). The administration of Abs against IL-10 in the PC + I/R group (PC + I/R + anti-IL-10) was also associated with IL-1β levels comparable to those observed after I/R. The effect of IL-10 on oxidative stress was evaluated. As shown in Fig. 6B, MDA levels after hepatic I/R showed only a slight tendency to be increased in normal livers; MDA levels in fatty livers were significantly higher after reperfusion. IL-1β seems to be involved in the increased oxidative stress seen in fatty livers after hepatic I/R because the IL-1 receptor antagonist pretreatment (I/R + IL-1ra) significantly reduced the increases in MDA levels. Exogenous IL-10 pretreatment, or preconditioning, which generated IL-10, reduced the increases in MDA found in fatty livers after I/R. Anti-IL-10 administration in the PC + I/R group (PC + I/R + anti-IL-10) resulted in MDA levels similar to those observed after I/R; IL-1 receptor antagonist supplementation (PC + I/R + anti-IL-10 + IL-1ra) led to oxidative stress values comparable to those obtained in the PC + I/R group.

Figure 6.

Effect of IL-10 on (A) IL-1β and (B) MDA levels at 6 hours of hepatic reperfusion. *P < .05 vs. Control. **P < 0.05 vs. I/R ***P < .05 vs. PC + I/R.

In accordance with the biochemical results, histological study in normal livers subjected to I/R showed, at 6 hours of reperfusion, moderate incipient hepatocyte necrosis and slight coagulative necrosis with neutrophil infiltration, randomly distributed throughout the hepatic parenchyma (Fig. 7A). However, multifocal areas of moderate coagulative necrosis with neutrophilic infiltration were observed in fatty livers (Fig. 7B). When preconditioning was performed, the extent of necrosis and number of necrotic areas were reduced in normal and fatty livers with respect to I/R. In normal livers (Fig. 7C), the histological study showed slight incipient and coagulative patchy necrosis of isolated hepatocytes distributed throughout the hepatic parenchyma; in fatty livers, patchy areas of hepatocyte incipient necrosis and scattered multifocal areas of coagulative necrosis were observed (Fig. 7D). At 24 hours of reperfusion, histological study of normal livers in I/R showed multifocal areas of moderate coagulative necrosis and neutrophil infiltration, randomly distributed throughout the parenchyma; in fatty livers, extensive and confluent areas of coagulative necrosis with neutrophil infiltration were observed. By contrast, preconditioning markedly reduced the extent of necrosis and number of necrotic areas at 24 hours after reperfusion in normal and fatty livers with respect to I/R. In normal liver, these areas were mainly of incipient necrosis; in fatty liver, patchy areas of hepatocyte incipient necrosis and scattered multifocal areas of coagulative hepatocyte necrosis were observed. At 6 hours (Fig. 7E to 7J) and 24 hours of reperfusion, the histological results corresponding to the PC + I/R + IL-1β (Fig. 7E and 7F) and PC + I/R + anti-IL-10 (Fig. 7G and 7H) groups were similar to those observed in the I/R group. The histological results corresponding to the PC + I/R + anti-IL-10 + IL-1ra (Fig. 7I and 7J), I/R + IL-1ra, and I/R + IL-10 groups were similar to those observed in the PC + I/R group.

Figure 7.

Histological lesions in liver at 6 hours of hepatic reperfusion. I/R (A. Ln, B. Ob). (A) Small area of coagulative hepatic necrosis (arrows) with neutrophil infiltration. (B) Widespread coagulative hepatic necrosis with neutrophil infiltration (arrows); PC +I/R (C. Ln, D. Ob). (C) Irregular area of incipient necrosis (asterisk). (D) Widespread coagulative hepatic necrosis with neutrophil infiltration (arrows), PC +I/R treated with IL-1β (10 and 20μg/kg in Ln and Ob, respectively) (E. Ln, F. Ob) and PC+I/R+anti-IL-10 (G. Ln, H. Ob): Similar to I/R; PC+I/R treated with anti-IL-10 (0.5 mg/kg) and IL-1ra (40 and 80 mg/kg in Ln and Ob, respectively) (I. Ln, J. Ob): Similar to PC +I/R. Hematoxilin-eosin; original magnification ×550.

With respect to survival experiments, all fatty rats died after 60 minutes of total hepatic ischemia within 3 days after liver surgery, and all lean animals survived the same ischemic challenge for 30 days after surgery. A significant improvement in survival was found in fatty rats subjected to hepatic ischemia with previous preconditioning or exogenous IL-10 pretreatment. Thus, 70% of animals with preconditioning and 60% of animals with IL-10 pretreatment survived for 30 days after surgery. IL-10 action inhibition in the preconditioned group reduced the benefits of preconditioning because 20% of animals survived for 30 days after surgery.

In order to determine the source of NO in preconditioning, we investigated the effect of NAME, versus a the effect of a selective iNOS inhibitor (AG). As shown in Fig. 8, NO synthesis inhibition with NAME in the preconditioned group (PC + I/R + NAME) eliminated the benefits of preconditioning, resulting in biochemical and histological parameters of hepatic injury similar to those observed in the I/R group. However, the inhibition of the iNOS activity in the preconditioned group (PC + I/R + AG) did not modify the benefits of preconditioning. The effects of L-NAME on IL-10, IL-1β and MDA levels were also investigated. As shown in Fig. 9, the inhibition of NO eliminated the benefits of preconditioning on IL-10 release. This was associated with IL-1β and MDA levels similar to those observed after hepatic I/R.

Figure 8.

Role of NO in steatotic livers undergoing hepatic I/R. (A) ALT levels in Ln and Ob at 6 hours of reperfusion. (B and C) Percentage of grade 3 necrosis in Ln and Ob at (B) 6 hours and (C) 24 hours of reperfusion. *P < .05 vs. Control. **P < .05 vs. I/R. ***P < .05 vs. PC + I/R.

Figure 9.

Effects of NO on (A) IL-10, (B) IL-1β, and (C) MDA levels at 6 hours of reperfusion. *P < .05 vs. Control. **P < .05 vs. I/R. ***P < .05 vs. PC + I/R.

Discussion

Higher levels of IL-1β were detected after I/R in fatty livers compared with normal livers. In addition, the increase in IL-10 levels detected in normal livers as a consequence of I/R was absent in fatty livers. This imbalance between pro- and anti-inflammatory ILs seen in steatotic livers subjected to I/R could explain, at least partially, the decreased tolerance of steatotic livers to this process. As in normal livers, pharmacological strategies consisting of inhibition of endogenous IL-1β action as well as exogenous IL-10 pretreatment could improve the tolerance of fatty livers to I/R injury and consequently decrease the inherent risk of liver surgery. However, potential difficulties for effectively preventing injury to normal and steatotic livers should not be discounted. The effective dose of these protective strategies may differ for the two types of livers, and the range in which these treatments are beneficial in hepatic I/R injury appears to be quite narrow.

The results of the current study indicate that the protection conferred by exogenous IL-10 against hepatic I/R injury was associated with reduction in IL-1β release. Thus, preconditioning, through IL-10 overproduction, could inhibit IL-1β release, thereby protecting normal and steatotic livers against hepatic I/R injury. The administration of anti-IL-10 Abs in the PC + I/R group (PC + I/R + anti-IL-10) eliminated the benefits of preconditioning on IL-1β levels and hepatic injury. However, the inhibition of IL-1β action in the PC + I/R + anti-IL-10 group (PC + I/R + anti-IL-10 + IL-1ra) resulted in biochemical and histological parameters of hepatic injury similar to those obtained when preconditioning was performed.

The effects of both IL-1 and IL-10 on oxidative stress have been previously reported: IL-1 is involved in hepatic-I/R-injury up-regulating reactive oxygen species (ROS) production.5, 32 Among the regulatory properties attributed to IL-10 is the inhibition of ROS production in cultured cells.33 Considering these observations on the effect of ILs on ROS production, and a recent study indicating that preconditioning reduces the increased lipid peroxidation observed in fatty livers as a consequence of I/R,15 we evaluated whether ischemic preconditioning, throughout IL-10 release, could reduce IL-1β–induced oxidative stress. Our results indicate that the administration of anti-IL-10 Abs in the PC + I/R group (PC + I/R + anti-IL-10), which eliminated the benefits of preconditioning on IL-1β levels and hepatic injury, resulted in oxidative stress values similar to those observed after hepatic I/R. However, the inhibition of IL-1β action in the PC + I/R + anti-IL-10 group (PC + I/R + anti-IL-10 + IL-1ra) did not modify the benefits of preconditioning on oxidative stress and hepatic injury.

Taking the results obtained into account, it appears unlikely that preconditioning could be dependent on iNOS. In addition, our previous studies on liver preconditioning performed in warm ischemia established that, immediately after preconditioning, tissue NO synthesis was increased.34, 35 Because there is clearly no time for the expression of the inducible form, this suggest that the newly synthesized NO originates from constitutive NO synthase (cNOS). Moreover, preconditioning does not promote any difference in iNOS with respect to the I/R group.34, 35

A recent study in myocardial ischemia indicates that NO is able to induce IL-10 production.19 The inhibition of IL-1 production by NO has been previously demonstrated in experimental models of I/R16, 17 and in cases of hepatotoxicity induced by LPS.18 The results of the present work seem to indicate a relationship between NO, IL-10 and IL-1β in hepatic I/R. The increase in IL-10 production induced by preconditioning could be mediated by NO. The inhibition of NO synthesis with NAME eliminated the benefits of preconditioning on IL-10 release because IL-10 levels were similar to those found after hepatic I/R. Because the IL-10 generated in preconditioning was able to inhibit IL-1β release, and consequently its injurious effects on hepatic I/R injury, the inhibition of NO synthesis in preconditioned animals resulted in hepatic IL-1β, oxidative stress, as well as biochemical and histological parameters of hepatic injury similar to those observed after hepatic I/R.

Data obtained after 90 minutes of partial hepatic ischemia (30%) indicated that preconditioning through NO mediation prevented the increase in tumor necrosis factor-α (TNF-α), thus attenuating the liver injury following I/R. The key role of TNF-α in hepatic I/R injury was confirmed by the use of antibodies against TNF-α.36 However, in the experimental model evaluated in the present study (60 minutes of 70% hepatic ischemia), the benefits of preconditioning could not be explained by a reduction in TNF-α release following I/R. Moreover, hepatic I/R was not associated with TNF-α production, indicating the minor role of this cytokine in hepatic I/R injury.15 Under these conditions, IL-1 seems to be involved in hepatic I/R injury. Preconditioning through NO mediation reduced IL-1β release and protected against hepatic I/R injury. These results contrast with the data reported in a previous study by Kato et al.37 in which IL-1 is not required for the hepatocellular injury induced by 90 minutes of partial ischemia (70%). As for TNF-α and IL-1β, the role of IL-10 may be different depending on the type and/or severity of I/R. Thus, the studies reported in the literature point to a beneficial effect of exogenous IL-10 in nonsteatotic livers undergoing hepatic I/R9–12 and a minor role of endogenous IL-10 in this process.38 However, our results suggest a beneficial role of both endogenous and exogenous IL-10 on hepatic I/R injury, at least under the conditions evaluated in the present study.

The molecular mechanisms of the modulatory effect of NO involved in ischemic preconditioning on IL-10 production have not been clarified in the present study. However, studies carried out to date point to the potential participation of nuclear factor-κB (NF-κB), activator protein 1 (AP-1) and p38 mitogen-activated protein kinase (p38 MAPK) in NO-induced IL-10 production. Thus, anti-inflammatory properties of NO in myocardial ischemia include IL-10 production through activation of gene transcription, which appears to be regulated by both NF-κB and AP-1 signaling pathways.19 Experimental data indicate that NO can induce preconditioning of hepatocytes by promoting the activation of p38 MAPK.39, 40 IL-10 production by p38 MAPK has been reported in LPS-stimulated macrophages.41, 42

Together, the results of the current work allow us to suggest a mechanism to explain the effects of preconditioning on IL release following hepatic I/R, summarized in Fig. 10. During hepatic I/R, steatotic livers showed lower IL-10 and higher IL-1β levels than in normal livers. Ischemic preconditioning increased IL-10 production, thus inhibiting IL-1β release and the ensuing hepatic injury that follows I/R in normal and steatotic livers. The effect of preconditioning on IL-10 production could be mediated by NO, probably originating from cNOS. These observations may be relevant to designing therapies in clinical practice that are aimed at protecting normal and steatotic livers against hepatic I/R injury.

Figure 10.

Schematic representation of the proposed mechanisms by which ischemic preconditioning modifies IL release following hepatic I/R.

Acknowledgements

The authors thank Emma Puig-Oriol and Llorenç Quintó (Epidemiology and Biostatistics Unit, Barcelona University), for their help in the statistical analyses. We acknowledge the generous supply of IL-1ra by Amgen Biologicals.

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