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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Ischemia/reperfusion (I/R) of the liver occurs in many clinical scenarios including trauma, elective surgery, and transplantation. Events initiated by this process can lead to inflammation in the liver, culminating in local injury as well as distant organ dysfunction. Recent studies have suggested that hypertonic saline exerts anti-inflammatory effects, which may be beneficial in preventing organ injury. In the present study, we examine the effect of hypertonic saline on the development of liver inflammation following I/R in both rat and mouse models. Hypertonic pretreatment was shown to prevent liver enzyme release concomitant with a reduction in liver neutrophil sequestration. Hypertonic saline appeared to exert this effect by inhibiting liver tumor necrosis factor α (TNF-α) generation, an effect that culminated in reduced liver adhesion molecule expression. Hypertonic saline pretreatment was shown to augment liver interleukin 10 (IL-10) expression following I/R, as a potential mechanism underlying its anti-inflammatory effect. To examine the role of IL-10 in the protective effect of hypertonic saline on liver I/R injury, we used a murine model of I/R. In wild type mice, hypertonic pretreatment similarly prevented liver injury induced by I/R. However, in IL-10 knockout animals, hypertonic pretreatment was unable to prevent the liver enzyme release, TNF-α generation, or neutrophil sequestration induced by I/R. In conclusion, these findings define a novel mechanism responsible for the anti-inflammatory effects of hypertonic saline and also suggest a potential clinical role for hyperosmolar solutions in the prevention of liver injury associated with I/R. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;40:211–220.)

Ischemia/reperfusion (I/R) injury of the liver is known to occur in many clinical scenarios, including major trauma with blood loss and extensive liver resections for tumor.1, 2 Experimental studies have defined a multistep process that culminates in the development of liver inflammation and hepatocellular necrosis. The initial phase involves activation of Kupffer cells with the production and release of reactive oxygen species, molecules that themselves exert hepatocellular injury.3 A second, later phase is characterized by further activation of Kupffer cells with the consequent release of inflammatory molecules that promote and amplify the development of hepatocellular injury.4 Central among these is the elaboration of the proinflammatory cytokine, tumor necrosis factor α (TNF-α).5, 6 TNF-α induces the upregulation of adhesion molecules including intercellular adhesion molecule 1 (ICAM-1) and vascular cellular adhesion molecule 1 (VCAM-1) as well as members of the selectin family.7, 8 TNF-α also contributes to an increase in liver C-X-C chemokine generation, which may further promote neutrophil recruitment and directed migration into the liver.4, 9, 10 Together, these events promote neutrophil sequestration into the sinusoids and subsequent transmigration into the hepatic parenchyma, the latter being a key element leading to hepatocellular injury. Finally, recent studies have reported that TNF-α–induced hepatocyte apoptosis is a critical early event in propagating the development of liver injury in models of I/R.11, 12 Strategies aimed at interfering with one or more of these processes have proven beneficial in minimizing experimental liver injury following I/R.13–15

Hypertonic solutions used as resuscitation fluids during critical illness have recently been shown to exert anti-inflammatory effects in vivo.16, 17In vitro studies have documented effects of hypertonic solutions on many cell types, including neutrophils, macrophages, and endothelial cells.18–21 We and others have reported the ability of hypertonic saline pretreatment to inhibit lipopolysaccharide (LPS)-induced release of TNF-α from macrophages in vitro.20, 22 This suggests that preconditioning with hypertonic solutions might preclude Kupffer cell TNF-α release and consequent liver injury. In the present study, we show that pretreatment with hypertonic saline prevents hepatocellular injury in two animal models of liver I/R. This appears to be due to the ability of hypertonic saline to prevent I/R-induced TNF-α production by the liver and hence precludes downstream events leading to neutrophil sequestration and injury, including up-regulation of endothelial cell adhesion molecule expression. Interestingly, hypertonic pretreatment also induces a significant increase in the production of the counterinflammatory cytokine interleukin 10 (IL-10). Studies using IL-10–deficient animals suggest that the ability of hypertonic pretreatment to induce IL-10 is central to the anti-inflammatory effects of this preconditioning strategy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Materials.

Guanidine isothiocyanate was obtained from Sigma (St. Louis, MO), Triton X-100 was obtained from Caledon (Georgetown, Canada), and 32P was obtained from Amersham (Oakville, Canada). Anesthetics used were ketamine obtained from Rogar/STB (London, Canada) and xylazine obtained from Bayer (Etobicoke, Canada).

Solutions.

Endotoxin-free Dulbecco's Modified Eagle Medium, Hank's balanced salt solution, phosphate-buffered saline, and RPMI were obtained from Life Technologies (Burlington, Canada). Polymorphonuclear leukocytes (PMNs) were isolated in Dulbecco's Modified Eagle Medium containing 10% fetal calf serum (HyClone, Logan, UT) and penicillin/streptomycin (Life Technologies). The University Health Network Pharmacy Department prepared sterile 7.5% NaCl for use in the studies.

Antibodies and Complementary DNA Probes.

Rabbit anti–rat IL-10 polyclonal antibody was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA); polyclonal rabbit anti–rat phospho-STAT3 (Tyr705) and polyclonal rabbit anti–rat STAT3 antibody were obtained from New England Biolabs (Beverly, MA); and murine monoclonal anti–human CD54 (ICAM-1) antibody was obtained from Zymed (San Francisco, CA). Murine ICAM-1 complementary DNA (cDNA), murine VCAM-1 cDNA, murine TNF-α cDNA and IL-10 cDNA probes were purchased from the American Type Culture Collection. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was obtained from Clontech (Palo Alto, CA).

Animals.

Adult male Sprague-Dawley rats weighing 300–350 g (Charles River, Canada) were allowed to acclimatize for 1 week. Rats were given standard laboratory rodent chow and water ad libidum. Twelve-week-old (28–30 g) male wild type (WT) mice (C56BL/6) or mice lacking expression of IL-10 on C57BL/6 background (B6.129P2(B6)-IL10tm1Cgn) were obtained from Jackson Laboratory (Bar Harbor, ME).

In Vivo Model of Warm Hepatic I/R Injury.

Rats were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (8 mg/kg). The right carotid artery was cannulated with a 22-gauge angiocath (Becton Dickinson, Franklin Lakes, NJ) for monitoring mean arterial pressure, blood sampling, and fluid administration. After a 10-minute stabilization period, animals received 4 mL/kg isotonic (0.9% NaCl, isotonic saline [ISO]) or hypertonic (7.5% NaCl, hypertonic saline [HTS]) saline via the arterial line. Sixty minutes later, rats underwent laparotomy. The portal venous and hepatic arterial branches to the left and median hepatic lobes were isolated and occluded with a 3.5-mm microvascular clamp. The incision was closed, and following a 30-minute ischemic period the abdomen was reopened and the clamp was removed, allowing reperfusion. Liver tissue was harvested at the indicated time point and frozen in liquid nitrogen for evaluation of myeloperoxidase levels, messenger RNA (mRNA), TNF-α or IL-10 or STAT protein, caspase 3, and nuclear factor κ B (NF-κB) activation.

The surgical procedure for I/R in the mouse was similar to that described by Lentsch and colleagues23 except that animals received 4 mL/kg volume of ISO (0.9%) or HTS (7.5%) via lateral tail vein at 60 minutes prior to the start of ischemia. After 45 minutes of partial hepatic ischemia, the clip was removed, initiating hepatic reperfusion. Mice were euthanized after indicated periods of reperfusion, and liver tissue and blood samples from cardiac puncture were recovered.

Assessment of Serum Sodium Concentration, Osmolarity, and Liver Enzyme Release.

Plasma was separated by centrifugation and osmolarity was measured in an Advanced Osmometer 3D3 (Two Technology Way, Norwood, MA). Plasma aspartate aminotransferase (AST) levels were measured at various times following reperfusion by the clinical biochemistry laboratory at the University Health Network.

Myeloperoxidase Assay.

Frozen liver samples were thawed, homogenized, and then freeze-thawed in phosphate-buffered saline containing 0.5% hexadecyltrimethylammonium bromide. The supernatant was used for myeloperoxidase (MPO) and protein assays. MPO activity was assessed through colorimetric assay using H2O2 as a substrate in a Cobas FARA II Chemistry System (Roche Diagnostic Systems, Nutley, NJ).24 The absorbance change per minute was used as a measure of MPO activity. Results are expressed as MPO activity/mg protein. Protein was measured using the Pierce bicinchoninic acid protein assay (Pierce, Rockford, IL).

RNA Extraction and Northern Blot Analysis.

Total RNA was extracted using the guanidium-isothiocyanate method.25 mRNA was isolated using an mRNA extraction kit (Quik Prep Micro Purification Kit, Amersham, Baie d'Urfe, Quebec). RNA was denatured, electrophoresed through a 1.2% formaldehyde-agarose gel, and transferred to a nylon membrane. Hybridization was performed using a 32P-labeled, random-primed murine ICAM-1 cDNA probe, murine VCAM-1 cDNA probe, murine TNF-α cDNA probe, or the murine IL-10 cDNA probe as described previously.26 Blots were washed under high stringency conditions, and specific mRNA bands were detected by autoradiography in the presence of intensifying screens. To control for loading, blots were stripped and reprobed for GAPDH. Densitometry was performed using the UN-SCAN-IT software (Silk-Scientific Corporation, Orem, UT) and a Hewlett-Packard 250L scanner (Hewlett-Packard, Palo Alto, CA). Segments were normalized against GAPDH values.

Nuclear Protein Extraction.

Nuclear protein extracts were prepared from aliquots of 200–500 mg of frozen liver tissue.27 Supernatants containing nuclear proteins were aliquoted into small fractions, frozen in liquid nitrogen, and stored at −70°C. Protein quantitation was performed using the BIORAD protein assay dye reagent (BIO-RAD, Hercules, CA).

Electrophoretic Mobility Shift Assay.

Electrophoretic mobility shift assay was used to assess NF-κB binding activity to the ICAM-1 variant NF-κB site. The probe used was a 25-bp double-stranded construct (5′-TAGCTTGGAAATTCCGGAGCTGAAG-3′) corresponding to a sequence in the ICAM-1 variant κB binding site.28 End labeling was performed by T4 kinase in the presence of [32P]ATP.

Western Blot Analysis.

To study ICAM-1 and TNF-α expression in the liver, whole liver was homogenized in Tris-buffered saline/1% Triton X-100 solution. Liver tissue homogenate (50 μg protein/sample) was separated on 15% SDS–polyacrylamide gel electrophoresis under nonreducing conditions.29 Separated proteins were electroblotted onto polyvinylidene difluoride membranes. After blocking, membranes were then incubated with a 1/1000 dilution of antiserum against the protein of interest at room temperature for 1 hour. Ag–Ab complexes were identified with goat anti–rabbit immunoglobulin G tagged with horseradish peroxidase (Sigma) and detected using the enhanced chemiluminesence detection system (Amersham, Arlington Heights, IL). TNF-α protein in serum and hepatic tissue homogenates (100 μL/sample) were evaluated using sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN).

Caspase 3.

Assay of caspase 3 activity was performed using a Caspase Assay Kit (Biosource, Camarillo, CA). Liver was homogenized and lysates were incubated with 25 μL of a specific substrate for caspase 3 (Ac-DEVD-pNA) in a 96-well plate. Following overnight incubation, plates were read using a colorimetric plate reader (Titertek Instruments, Inc., Huntsville, AL) at 409 nm.30

Statistics.

Data are presented as the mean ± SE of the number of experiments indicated. Significance was assessed using one-way ANOVA with post hoc testing with the Tukey-Kramer multiple comparisons test. P < .05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Animal Welfare, Mean Arterial Pressure, and Changes in Serum Sodium and Osmolality.

Animals in all groups tolerated the surgical manipulations and recovered well from their general anaesthesia. Mean arterial pressure was measured to exclude any gross differences in animals' macrohemodynamic state that might account for any differential effects of HTS administration on liver injury (Fig. 1A). There were no significant differences in mean arterial pressure among groups.

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Figure 1. Effect of pretreatment with isotonic or hypertonic solution on mean arterial pressure, serum sodium, and osmolality. Animals were sequentially evaluated for (A) mean arterial pressure, and (B) serum osmolality at various time points after treatment with the isotonic or hypertonic preconditioning regimens. The data represent the mean ± SEM for at least three animals per group for each time point. *P < .05 versus isotonic at same time point. **P < .01 versus isotonic at same time point. MAP, mean arterial pressure.

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Serum sodium (Supplemental Fig. 1)and osmolality (Fig. 1B) were measured to discern the effects of HTS or ISO pretreatment. Baseline plasma sodium was 142 ± 2 meq/L. Serum sodium peaked in HTS animals at 10 minutes after HTS (158 ± 5 meq/L) and declined thereafter; however, it remained elevated compared with isotonic animals for the duration of the experimental protocol (P < .05). Serum osmolality was 302 ± 2 mosmol/kg H2O in control rats. Following HTS, serum osmolality peaked within 5 minutes of HTS bolus at 337 ± 3 mosmol/kg H2O and declined toward normal over 30 minutes.

Effect of Hypertonic Pretreatment on Liver AST Release.

In our previous studies, HTS administration in advance of the beginning of the ischemic period resulted in protection against AST release measured 4 hours after clamp release and the initiation of reperfusion.21 To determine the effect of timing of HTS administration on the HTS protection from I/R injury, rats received HTS at various time points relative to the initiation of reperfusion (i.e., clamp release). These included animals receiving HTS at the time of hepatic reperfusion (t = 0 hours relative to reperfusion), or 2 hours after reperfusion had occurred (t = 2 hours relative to reperfusion). Plasma AST levels were then assessed at the 4 hour reperfusion time point as an index of liver injury (Supplemental Fig. 2). As shown, I/R following isotonic saline pretreatment caused a 4.5-fold increase in AST release compared with sham-operated animals, while in animals pretreated with HTS 1 hour prior to the initiation of hepatic ischemia (t = −1.5 hours relative to reperfusion), AST increases were abrogated. However, the protective effect of HTS was lost when HTS was administered at the time of reperfusion (t = 0 hours relative to reperfusion) or 2 hours after reperfusion had occurred (t = 2 hours relative to reperfusion). These results suggest that the timing of HTS administration is critical in determining its protective effect on hepatic I/R injury.

Effects of HTS on Liver Neutrophil Accumulation.

To determine if HTS pretreatment was accompanied by decreased PMN sequestration, we measured liver MPO, a PMN marker enzyme (Fig. 2A). I/R in isotonic-pretreated animals caused a fivefold increase in MPO compared with sham-operated animals. However, HTS protected against I/R-induced hepatic PMN sequestration with 4-hour reperfusion, showing MPO levels that were comparable with control.

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Figure 2. Effect of hypertonic pretreatment on neutrophil sequestration and adhesion molecule expression in the liver. Animals were subjected to I/R following pretreatment with either ISO or HTS. (A) Effect of HTS on total liver MPO determined at 4 hours of reperfusion. The data represent the mean ± SEM for at least three animals per study. *P < .05 versus ISO. (B) Effect of pretreatment with either ISO or HTS on levels of ICAM-1 mRNA in the liver at 30 minutes of reperfusion (upper two panels) and 4 hours of reperfusion (lower two panels). For each, a representative blot is shown on the left and densitometry of ICAM-1 mRNA levels normalized to GAPDH. For densitometry, the data represent the mean ± SEM for at least three animals per study. *P < .05 versus ISO. (C) Effect of pretreatment on levels of VCAM-1 mRNA in the liver at 30 minutes of reperfusion (upper two panels) and 4 hours of reperfusion (lower two panels). For each, a representative blot is shown on the left and densitometry of VCAM-1 mRNA levels normalized to GAPDH. The data represent the mean ± SEM for at least three animals per study. *P < .05 versus ISO. (D) Electrophoretic mobility shift assay examining the effect of ISO or HTS pretreatment on NF-κB translocation in the liver at various time points following the initiation of reperfusion. The data are representative of three separate studies. The cold lane represents the use of excessive unlabelled probe using the t = 120 minute time point. MPO, myeloperoxidase; ISO, isotonic saline; HTS, hypertonic saline; I/R, ischemia/reperfusion; ICAM-1, intercellular adhesion molecule 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VCAM-1, vascular cellular adhesion molecule 1; NF-κB, nuclear factor κ B.

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ICAM-1 and VCAM-1 are important in the adherence, transmigration, and adhesion-dependent cytotoxicity of PMNs in hepatic I/R.14, 15 We examined the effect of HTS on the expression of hepatic ICAM-1 mRNA at 30 minutes and 4 hours reperfusion (Fig. 2B, upper and lower panels, respectively). Sham animals did not exhibit ICAM-1 expression. ICAM-1 mRNA expression was significantly increased following I/R at both the early and later time points in animals receiving isotonic pretreatment fluids. HTS pretreatment completely prevented early I/R-induced ICAM-1 mRNA and significantly reduced its expression at 4 hours reperfusion (see Fig. 2B). Similar effects of HTS on VCAM-1 mRNA were observed (Fig. 2C, upper and lower panels, respectively).

NF-κB binding to the ICAM-1 promoter following reperfusion was evaluated to determine if HTS pretreatment modulates the activation of this intracellular signaling pathway (Fig. 2D).31 Reperfusion of isotonic I/R animals increased NF-κB binding to the ICAM-1 promoter. In contrast, I/R-induced NF-κB binding to the ICAM-1 promoter was prevented by hypertonic pretreatment. NF-κB translocation was observed when hypertonic saline was given at the beginning of reperfusion, consistent with the lack of inhibition by hypertonic treatment when administered at this time (data not shown). These results suggest an ability of HTS to modulate the activation of intracellular signaling pathways is responsible for reduced hepatic ICAM-1 adhesion molecule expression.

Consistent with changes in ICAM-1 gene expression, HTS pretreatment also prevented the I/R-induced rise in liver ICAM-1 protein levels (Supplemental Fig. 3).

Effects on Hepatocellular Apoptosis.

Recent studies have suggested a role for hepatocellular apoptosis in liver I/R injury.11 Caspase 3 levels were measured in liver tissue following I/R in animals with isotonic or hypertonic pretreatment. As shown in Fig. 3, I/R itself induced activation of the enzyme caspase 3 compared with sham animals. A further significant rise in caspase 3 activity was observed in I/R animals pretreated with hypertonic saline.

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Figure 3. The effect of ISO or HTS pretreatment on hepatocellular caspase 3 activity following I/R. The liver samples are taken at 4 hours following the beginning of reperfusion and the data represent the mean and standard error of 3 animals per group. **P < .01 versus ISO. CTRL, control; ISO, isotonic saline; HTS, hypertonic saline.

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Altered TNF-α Expression Following HTS Pretreatment.

To determine if HTS pretreatment modulates TNF-α expression following hepatic I/R as a mechanism leading to reduced hepatocellular injury, TNF-α mRNA levels in liver samples were assessed at 30 minutes of reperfusion. Figure 4A shows that TNF-α mRNA expression was minimal in the sham group, while I/R induced a marked increase in TNF-α mRNA levels at the 30-minute reperfusion time point in ISO animals. By contrast, HTS pretreatment reduced I/R-induced TNF-α mRNA expression at 30 minutes compared with isotonic animals (see Fig. 4A).

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Figure 4. Effect of hypertonic pretreatment on TNF-α levels. (A) Effect of I/R on liver TNF-α mRNA levels at 30 minutes of reperfusion following pretreatment with either ISO or HTS. A representative blot is shown on the left and densitometry of TNF-α mRNA levels normalized to GAPDH is presented on the right. The data represent the mean ± SEM for at least three animals per study. *P < .05 versus ISO. (B) TNF-α protein levels in the liver at 2 hours of reperfusion. Animals were pretreated with ISO or HTS and then exposed to I/R. At 2 hours, liver was recovered for measurement of TNF-α protein. The data represent the mean ± SEM for at least three animals/study. *P < .05 versus ISO. I/R, ischemia/reperfusion; ISO, isotonic saline; HTS, hypertonic saline; TNF-α, tumor necrosis factor α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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TNF-α protein expression in liver was assessed at the 2-hour reperfusion time point (Fig. 4B). I/R significantly increased hepatic TNF-α protein levels in isotonic pretreated rats. Consistent with mRNA levels, HTS pretreatment prevented the I/R-induced increase in TNF-α protein observed in isotonic I/R animals. These results support the concept that HTS preconditioning is able to prevent I/R-induced liver injury by reducing TNF-α generation and thus minimizing its downstream effects.

IL-10 Expression Following Liver I/R.

IL-10 has been shown to suppress the production of proinflammatory molecules such as TNF-α32 and to inhibit liver I/R injury when administered exogenously.13 We evaluated IL-10 expression in I/R animals and found that I/R caused a modest increase in liver IL-10 mRNA levels compared with sham animals by 30 minutes of reperfusion (Fig. 5A). By contrast, hypertonic treatment prior to I/R caused a marked rise in IL-10 mRNA levels compared with I/R animals receiving ISO pretreatment. Hypertonic treatment without I/R did not affect levels of IL-10 expression (data not shown). Consistent with the effects on IL-10 mRNA levels, hypertonic pretreatment was observed to increase liver IL-10 protein expression compared with isotonic pretreated animals at 60 minutes of reperfusion (Fig. 5B). Indeed, IL-10 levels in the liver from I/R animals pretreated with hypertonic saline were found to be elevated at the beginning of reperfusion and progressively increased to the 60-minute postreperfusion time point (Fig. 5C).

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Figure 5. Effect of pretreatment on IL-10 expression. (A) Effect of pretreatment with either ISO or HTS on levels of IL-10 mRNA in the liver at 30 minutes of reperfusion. A representative blot is shown of three animal studies. (B) IL-10 protein levels in the liver at 1 hour of reperfusion. Animals were pretreated with ISO or HTS and then exposed to I/R. At 1 hour, liver was recovered for measurement of IL-10 protein by Western blot analysis. The blot is representative of three separate studies. (C) IL-10 protein levels in the liver at various time points beginning with the start of reperfusion (t = 0 hours). Animals were pretreated with HTS and then exposed to ischemia. Liver was recovered for measurement of IL-10 protein by Western blot analysis at the onset of reperfusion and at subsequent noted time points. The blot is representative of two separate studies. (D) Levels of STAT3 and phosphorylated STAT3 at various time points after the initiation of reperfusion following pretreatment with either ISO or HTS. The blot is representative of three separate studies. I/R, ischemia/reperfusion; ISO, isotonic saline; HTS, hypertonic saline; IL-10, interleukin 10; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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IL-10 is predominantly regulated at the level of transcription. Several transcription factors contribute to IL-10 transcription.33, 34 Among these, STAT3 has been shown to be an important regulator of the IL-10 gene.33 Phosphorylation of STAT3 was measured as evidence of its activation. In contrast to sham animals or ISO I/R animals, there was evidence of STAT3 phosphorylation at the end of the ischemic phase in cells pretreated with HTS (Fig. 5D). By 90 minutes of reperfusion, there was a marked increase in STAT3 phosphorylation in the HTS-pretreatment group compared with ISO I/R or sham animals. Although there was some variability in total STAT3 loading, this did not appear to account for the increase (Fig. 5D). Considered together, these studies indicate that HTS pretreatment accentuates I/R-induced IL-10 gene and protein expression in the liver and induces activation of at least one transcription factor implicated in the induction of IL-10 gene expression.

To directly evaluate the role of IL-10 in the protective effect of HTS on liver injury following I/R, we evaluated liver I/R in the mouse to study IL-10 knockout (KO) animals. As illustrated in Fig. 6A, a 45-minute ischemic period followed by reperfusion caused a progressive rise in AST levels in WT animals. At both 60 minutes and 3 hours of reperfusion, HTS pretreatment caused a significant reduction in AST release compared with ISO-pretreated mice. IL-10 KO mice appeared more susceptible to hepatocellular injury following I/R with AST levels at both 60 minutes and 3 hours following reperfusion compared with WT animals (compare ISO-treated I/R animals with ISO-treated IL-10 KO I/R animals). In contrast to WT animals, however, HTS pretreatment did not exert a protective effect on liver injury in IL-10 KO animals.

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Figure 6. Effect of hypertonic pretreatment on liver inflammation following I/R in WT and IL-10 KO animals. (A) Effect of ISO or HTS pretreatment on serum AST levels at t = 1 and 3 hours following reperfusion in WT and KO animals. *P < .05 versus respective WT or KO sham animals. **P < .05 versus WT ISO pretreatment animals. Black bars are from animals pretreated with ISO and gray bars are from animals pretreated with HTS. The data are the mean ± SE from three to six animals per group. (B) Effect of ISO or HTS pretreatment on liver MPO levels at t = 1 hour following reperfusion in WT and KO animals. *P < 0.05 versus sham animals. **P < .05 versus ISO pretreatment animals. Black bars are from animals pretreated with ISO and gray bars are from animals pretreated with HTS. The data are the mean ± SE from three to six animals per group. (C) Effect of ISO or HTS pretreatment on serum TNF-α levels at t = 1 hour following reperfusion in WT and KO animals. *P < .05 versus sham animals. **P < .05 versus ISO pretreatment. Black bars are from animals pretreated with ISO and gray bars are from animals pretreated with HTS. The data are the mean ± SE from three to six animals per group. I/R, ischemia/reperfusion; AST, aspartate aminotransferase; WT, wild type; KO, knockout; MPO, myeloperoxidase; TNF-α, tumor necrosis factor α.

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The failure of HTS pretreatment to exert anti-inflammatory effects in IL-10 KO animals was demonstrated using other markers. The ability of HTS to prevent neutrophil sequestration in the liver in WT animals as evidenced by a reduction in MPO levels was not observed in IL-10 KO animals (Fig. 6B). Similarly, at 3 hours of reperfusion, HTS pretreatment reduced serum TNF-α by ≈70% in WT animals, while having no protective effects in IL-10 KO animals (Fig. 6C). For both parameters, the overall inflammatory response in IL-10 KO animals following I/R was augmented.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The present study demonstrates that pretreatment of animals with hypertonic saline reduces hepatocellular injury induced by I/R. The data suggest that this effect is due to the ability of HTS pretreatment to inhibit TNF-α production in vivo and thereby preclude downstream effects such as adhesion molecule expression and consequent neutrophil sequestration. A direct effect of HTS on endothelial cell adhesion molecule expression may be contributory but is unlikely to be central to the protective effect. We have previously reported that HTS has only a small direct effect on LPS-induced endothelial cell ICAM-1 expression, and only at osmolarity levels not usually achievable in the blood in vivo (≈450–500 mOsM).21 Considered together, HTS protection in this model appears to be due to inhibited TNF-α release following I/R.

Other investigators have reported a direct inhibitory effect of HTS on LPS-induced signaling leading to impaired TNF-α release by macrophages.20 A central novel finding of the present study is that HTS impairs TNF-α generation by the liver following I/R by inducing expression of the counterinflammatory cytokine IL-10. The latter assertion is supported by studies in IL-10 KO animals wherein HTS failed to prevent I/R injury in parallel with the restoration of liver TNF release and neutrophil sequestration. The mechanism whereby IL-10 inhibits TNF release was not directly examined in the present studies, although previous reports have demonstrated a direct effect on cell signaling.35 One recent report suggested that IL-10 might exert its inhibitory effect on TNF release through induction of heme oxygenase and consequent carbon monoxide generation.36 Further studies examining the contributions of IL-10–induced heme oxygenase expression in this model are warranted.

The cell of origin of the enhanced IL-10 production was not investigated in the present study. One likely candidate cell is the Kupffer cell, which is known to produce IL-10 in response to LPS stimulation.37 In this regard, we previously reported that HTS augmented LPS-induced IL-10 production by murine peritoneal macrophages in vitro.21 Strategies aimed at neutralizing IL-10 failed to alter LPS-induced cellular activation in that model, suggesting that IL-10 was not solely responsible for the observed inhibitory effect of HTS. This does not rule out the possibility that Kupffer cells may be the major source of IL-10 in the present in vivo I/R model, because these cells are known to have phenotypic differences from peritoneal macrophages, despite their common lineage. Furthermore, Kupffer cells may require cell–cell interactions in vivo to optimally produce IL-10. Finally, other cells in the liver—possibly parenchymal cells—might be important. In this regard, Lee and Song recently reported that liver fragments following I/R were able to exert tumor cell cytotoxicity when derived from IL-10 +/+ animals but not IL-10 −/− animals.38 Although the precise source of IL-10 was not noted in the preparation, it likely consisted predominantly of parenchymal cells, because no enrichment strategy was used.

Apoptosis has been implicated as an important mechanism contributing to liver inflammation and injury following I/R.11, 12 Consistent with this, I/R of the liver following isotonic pretreatment resulted in augmented caspase 3 activity. Hypertonic treatment prior to I/R further augmented caspase 3 activity. The activation of caspase 3 following hypertonic pretreatment is consistent with the report by Reinehr and colleagues, showing that hyperosmolarity sensitized hepatocytes for induction of apoptosis by CD95 ligand.39 In addition, the HTS-induced impairment of NF-κB translocation would have removed a major antiapoptotic signal in the liver.40 Considered together, it appears unlikely that the protective effect of hypertonic pretreatment was due to inhibition of hepatocyte apoptosis.

Corso and colleagues have previously demonstrated that fluid resuscitation with HTS/6% dextran solution attenuated leukocyte sequestration in the sinusoids and postsinusoidal venules in a model of shock/resuscitation in rodents.41, 42 A salutory effect of this strategy on hepatocellular injury was not demonstrated, although bile flow and hepatic energetic status were preserved. These investigators suggested effects of hypertonic saline/dextran on neutrophil–endothelial cell interactions in the liver as well as on neutrophil rigidity leading to trapping of cells and impediment of microvascular flow. In these studies, dextran alone had comparable effects to HTS/dextran. This suggests the possibility that the effect of HTS/dextran may have, in part, been related to other properties of dextran such as oxygen scavenging and coating of adhesive surface structures on the endothelium.43

Our recent studies have suggested one intriguing mechanism whereby hypertonicity might contribute to the induction of IL-10. This work reported that hyperosmolarity was able to activate the nonreceptor protein tyrosine kinase Fer with consequent activation of various downstream events.44 One such event is the phosphorylation and activation of STAT3, a transcription factor known to be involved in IL-10 gene transcription.45 Consistent with this notion, HTS was shown to augment STAT3 phosphorylation. Because both phosphorylated STAT3 and IL-10 protein were detected in the liver at the onset of reperfusion, it is also possible that the phosphorylation of STAT3 is a downstream effect of the IL-10 receptor activation by the newly synthesized IL-10. Further studies examining events during the ischemic phase of liver I/R will help to unravel the temporal relationship of these events. Nevertheless, this might serve to further augment the inhibitory effects of hypertonic treatment, possibly through the induction of genes such as suppressor of cytokine signaling 3.46

Recent studies in humans have shown that preconditioning of the liver with a transient period of warm ischemia prevented subsequent I/R injury during liver surgery.47 The present study suggests that pretreatment with hypertonic solutions might similarly exert hepatoprotective effects in humans. The safety and ease of HTS infusion make it a clinically desirable preventative intervention. In addition, the mechanism described herein—namely induction of a counterinflammatory response by hypertonic exposure—may be relevant to the prevention of I/R damage in other organ systems. Further studies to evaluate the role of hypertonic preconditioning as a means of minimizing organ injury in various experimental and clinical settings appear warranted.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
  • 1
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    Heckbert SR, Vedder NB, Hoffman W, Winn RK, Hudson LD, Jurkovich GJ, et al. Outcome after hemorrhagic shock in trauma patients. J Trauma 1998; 45: 545549.
  • 3
    Jaeschke H, Farhood A. Neutrophil and Kupffer cell-induced oxidant stress and ischemia- reperfusion injury in rat liver. Am J Physiol 1991; 260: G355G362.
  • 4
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

FilenameFormatSizeDescription
suppmat_211_fig1.ppt43KEffect of pretreatment with isotonic or hypertonic solution on mean arterial pressure, serum sodium and osmolality. Animals were sequentially evaluated for serum sodium at various time points after treatment with the isotonic or hypertonic preconditioning regimens.

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