Synergistic induction of IL-10 by hypertonic saline solution and lipopolysaccharides in murine peritoneal macrophages

Synergistic induction of IL-10 by hypertonic saline solution and lipopolysaccharides in murine peritoneal macrophages George D. Oreopoulos, MD, Suzanne Bradwell, Ziyue Lu, MD, Jie Fan, PhD, Rachel Khadaroo, MD, John C. Marshall, MD, Yue Hua Li, MD, and Ori D. Rotstein, MD, Toronto, Ontario, Canada

Background. Liver injury after ischemia/reperfusion is an important cause of morbidity in surgical patients. We have shown that the preconditioning of animals that were subjected to liver ischemia/reperfusion with hypertonic saline solution (HTS) prevented injury by inhibiting Kupffer cell tumor necrosis factor (TNF) production. We postulated that the induction of anti-inflammatory interleukin-10 (IL-10) by HTS might contribute to protection. Methods. Murine thioglycolate–elicited peritoneal exudative macrophages (PEMs) were used to model the effects of HTS on IL-10 release from Kupffer cells. Cells were preconditioned with 500 mOsm HTS (or isotonic saline medium) for 2 hours and then stimulated with lipopolysaccharide (LPS; 1 µg/mL) or vehicle for 4 hours under isotonic conditions. TNF-α and IL-10 were measured in the culture supernatant by enzyme-linked immunosorbent assay; TNF, IL-10, and SOCS-3 messenger RNA expression were assessed by Northern blot. NF-κB activation was examined by electrophoretic mobility shift assay and Western blot for IκB degradation. Results. In the absence of LPS, isotonic medium–and HTS-pretreated PEMs produced little IL-10 (24.9 ± 66.0 and 0 pg/mL, respectively); however, stimulation of PEMs with LPS increased IL-10 (134.9 ± 72.2 pg/mL). Preconditioning with HTS significantly augmented LPS-induced IL-10 production, resulting in a 2-fold increase in IL-10 compared with the isotonic solution LPS group (270.7 ± 106.8 pg/mL; P < .01). HTS alone increased IL-10 mRNA levels and markedly augmented levels induced by LPS alone. To determine whether IL-10 accounted for HTS-induced TNF inhibition, cells from IL-10 knockout animals were studied. A lack of IL-10 did not reverse the inhibitory effect of HTS on LPSinduced TNF. NF-κB activation was the same in HTS-and isotonic solution–pretreated groups after LPS. Conclusions. HTS augments IL-10 induction by LPS at the gene level. Although TNF is reduced, it is not causally related to increased IL-10 or altered NF-κB signaling. HTS might exert its beneficial effects by independently modulating pro- and anti-inflammatory molecules, accounting for the potent immunomodulation exerted by HTS in vivo. (Surgery 2001;130:157-65.) From the Departments of Surgery, University Health Network, and University of Toronto, Toronto, Ontario, Canada

A NUMBER OF CLINICAL TRIALS have demonstrated the safety and efficacy of hypertonic saline solution (HTS) resuscitation for patients in hemorrhagic shock.1-4 In addition to the known ability of HTS to effect rapid restoration of intravascular volume Presented at the 62nd Annual Meeting of the Society of University Surgeons, Chicago, Ill, February 8-10, 2001. Reprint requests: Ori D. Rotstein, MD, Toronto General Hospital, 200 Elizabeth St, EN9-232, Toronto, Ontario, Canada, M5G 2C4. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 11/6/115829 doi:10.1067/msy.2001.115829

and to improve myocardial contractility, uncontrolled studies that used HTS have repeatedly noted a trend towards a reduced incidence of systemic complications after resuscitated shock, a form of global ischemia-reperfusion (I/R) injury.3,5 We reported a rodent model of posthemorrhage lung injury in which rats that were resuscitated with HTS exhibited less lung injury compared with rats that received isotonic resuscitation fluids.6 Although the mechanism is poorly defined, it has been suggested that reduced neutrophilendothelial cell interactions contribute to these effects.6-9 Our group subsequently reported on the SURGERY 157

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Fig 1. Hypertonic modulation of LPS-induced PEM TNF-α and IL-10. PEMs were incubated in the presence of LPS (1 µg/mL) for 4 hours under isotonic conditions with or without pretreatment with 500 mOsm HTS for 2 hours. TNF-α and IL-10 (A and B, respectively) were measured in the culture supernatants by ELISA at the end of the LPS incubation period. The groups included unmanipulated control cells (Control), PEMs exposed to hypertonicity for 2 hours and then resuspended in isotonic medium alone (HTS), PEMs stimulated with LPS for 4 hours under isotonic conditions (LPS), and cells that were preconditioned with HTS for 2 hours and then resuspended in isotonic medium and stimulated with LPS for 4 hours (HTS-LPS). *P < .05 vs LPS; **P < .01 vs LPS; ***P < .001 vs LPS.

ability of HTS infusion to protect against I/R injury to the liver.10 HTS administration decreased liver injury during the early, macrophage-mediated reperfusion phase.10 This protective effect was accompanied by reduced hepatic expression of the endothelial cell adhesion molecule intercellular adhesion molecule–1 (ICAM-1). Furthermore, preconditioning endothelial cells in vitro with HTS prevented their subsequent expression of ICAM-1 after activation with an inflammatory stimulus (lipopolysaccharide [LPS]). These results suggest that previous exposure of cells to hypertonic conditions (hypertonic preconditioning) might be able to modulate their response to a subsequent insult with beneficial effects at the whole organ level. Macrophages regulate many aspects of the pathogenesis of the acute respiratory distress syndrome, hepatic I/R, and other acute inflammatory states. Cells of the monocyte/macrophage lineage have both a protective role and the potential to cause host tissue injury. Activated macrophages modulate the activity of neighboring cells in a paracrine fashion by releasing proinflammatory cytokines, such as tumor necrosis factor–α (TNFα).11 The inhibition of macrophage activity indirectly modulates polymorphonuclear accumulation at sites of inflammation by reducing adhesion molecule and chemokine expression.12 Our previous studies in vivo demonstrated that HTS modulates I/R-induced pro- and anti-inflammatory cytokine expression at the whole organ level. I/R-induced

hepatic TNF-α expression was prevented and was accompanied by the augmented expression of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Because macrophages represent a major source of these cytokines in acute lung injury and hepatic I/R, we modeled the effects of HTS on these cells in vivo by examining the effects of HTS on peritoneal macrophage function in vitro. Using these cells, we examined the regulation of cytokine expression in these cells and evaluated the mechanisms whereby HTS might modulate the balance between pro- and anti-inflammatory cytokine induction. In these studies, we demonstrate that HTS pretreatment inhibits LPS-induced TNF protein and messenger RNA (mRNA) expression. This effect appears independent of LPS-stimulated NFκB nuclear translocation. Further, the data suggest that, although HTS augments LPS-induced IL-10 expression, the enhanced IL-10 release is not responsible for the inhibition of TNF release. Considered together, these findings demonstrate the profound effects of HTS pretreatment on the regulation of the inflammatory response. MATERIAL AND METHODS Material. Triton X-100 was purchased from Caledon (Georgetown, Canada), and 32P was purchased from Amersham (Oakville, Canada). EDTA and NaCl were obtained from BDH (Toronto, Canada). LPS (Escherichia coli 0111:B4) was obtained from Sigma (St Louis, Mo). Guanidine

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Fig 2. Northern blot analysis for PEM TNF-α and IL-10 mRNA expression. Treatment groups included control cells (CTRL), isotonic-LPS (LPS), hypertonic pretreatment for 2 hours followed by isotonic medium alone (HTS), and hypertonic pretreatment for 2 hours followed by LPS exposure under isotonic conditions for 1 hour (HTS + LPS). Lower panel shows Northern blot for GAPDH to control for sample loading.

isothiocyanate, 3% thioglycolate and LPS-free Dulbecco’s modified Eagle medium, Hanks’ balanced salt solution, phosphate-buffered saline solution, and Roswell Park Memorial Institute medium (RPMI) were obtained from Life Technologies (Burlington, Canada). Anti–IL-10 neutralizing antibody (AF519) was purchased from R&D Systems (Minneapolis, Minn). The IL-10 receptor antagonist, 1B1.2, was a gift from Dr Alice Mui (University of British Columbia, Vancouver, Canada). Animals. Animals were cared for in accordance with the guidelines set forth by the Canadian Council on Animal Care; the experimental protocol underwent previous review and approval by the University Health Network Animal Care Committee. Female BALB-c mice aged 6 to 7 weeks were obtained from Taconic Farms, fed a normal diet, and allowed to acclimatize for 1 week before use. C57BL/10-Il10tm1Cgn mice were obtained from the University of Cologne, Germany, through Jackson Laboratories and housed under pathogenfree conditions. Peritoneal exudative macrophages. Peritoneal exudative macrophages (PEMs) were harvested in ice-cold Hanks’ balanced salt solution 5 to 6 days after the intraperitoneal injection of 2 mL of sterile thioglycolate. PEMs were washed and resuspended in RPMI, 10% fetal calf serum, L-Gln at 1 × 106 cells/mL. This procedure consistently yields more than a 96% macrophage cell population by Wright’s stain, with more than 97% viability by trypan blue exclusion. Cells were incubated for 60 minutes at 37°C, 5% carbon dioxide before experimentation. PEMs were then incubated at 37°C in 5% carbon dioxide for 2 hours in either isotonic (290 mOsm) medium (RPMI, 10% fetal calf serum, L-Gln at 1 × 106 cells/mL) or medium that had been made hypertonic (500 mOsm) by the addi-

tion of sterile NaCl before the activation with LPS (1 µg/mL) under isotonic conditions. Measurement of TNF-α and IL-10. TNF-α and IL10 proteins in culture supernatants were evaluated by enzyme-linked immunosorbent assay (ELISA; R&D Systems) 240 minutes after LPS was added to cell suspensions with or without HTS pretreatment and compared with standard curves. RNA extraction and Northern blot analysis. Total RNA was extracted from PEMs with the use of the guanidinium-isothiocyanate method.13 Briefly, 10 × 106 PEMs were harvested and resuspended in 1.5 mL of 4 mol/L guanidine-isothiocyanate containing 25 mmol/L sodium citrate, 0.5% sarcosyl, and 100 mmol/L β-mercaptoethanol. mRNA was isolated with the use of an mRNA extraction kit (Quik Prep Micro Purification Kit; Amersham Pharmacia Biotech, Inc, Baie d’Urfe, Quebec), denatured, and electrophoresed through a 1.2% formaldehyde-agarose gel followed by transfer to a nylon membrane (NEN Life Sciences, Boston, Mass). Hybridization was performed with the use of 32P-labeled, random-primed murine TNF-α, IL-10 complementary DNA (cDNA) probes (American Type Culture Collection, Manassas, Va), or a SOCS3 cDNA probe (Dr Alice Mui, University of British Columbia, Vancouver, Canada). Blots were then washed, and specific mRNA bands were detected by autoradiography. Loading was controlled for by stripping blots and reprobing membranes with a G3PDH cDNA probe (Clontech, Palo Alto, Calif). Nuclear protein extraction and electrophoretic mobility shift assay. Nuclear protein extracts were prepared from 15 × 106 cells/group as described previously.14 PEMs were resuspended in 1 mL of solution A (150 mmol/L NaCl, 10 mmol/L HEPES pH 7.9, 1 mmol/L EDTA, and 0.5 mmol/L PMSF). The cells were centrifuged at 2000 rpm for 5 minutes and resuspended in solution A + 0.1% NP-40. Cells

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Fig 3. Northern blot analysis for PEM SOCS-3 mRNA expression. Treatment groups included control cells (CTRL), isotonic-LPS (LPS), hypertonic pretreatment for 2 hours followed by isotonic medium alone (HTS), and hypertonic pretreatment for 2 hours followed by LPS exposure under isotonic conditions (HTS + LPS). Lower panel shows Northern blot for GAPDH to control for sample loading.

were incubated on ice for 10 minutes, vortexed gently, and centrifuged at 13,000 rpm for 10 minutes. The supernatant was collected for analysis of cytoplasmic extracts by Western blot for IκB protein. Nuclear pellets were resuspended in 30 µL of solution B (25% glycerol, 20 mmol/L HEPES, pH 7.9, 420 mmol/L NaCl, 1.2 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, 2 mmol/L benzamidine, 5 µg/mL pepstatin, 5 µg/mL leupeptin, and 5 µg/mL aprotinin) and incubated on ice for 15 minutes. Nuclei were then obtained by centrifugation at 14,000 rpm, and proteins were measured with the BIORAD protein assay dye reagent (BIO-RAD, Hercules, Calif). Electrophoretic mobility shift assay (EMSA) was used to assess NF-κB binding activity to the κB binding site in the HIV-1 enhancer region (5´- AGG GAC TTT CCG CTG GGA CTT TCC –3´). End labeling was performed by T4 kinase in the presence of [32P]ATP. Five micrograms of nuclear protein was incubated with the labeled probe in the presence of 5 µg of nonspecific blocker, poly(dI-dC) in binding buffer (10 mmol/L Tris-HCL, pH 7.5, 100 mmol/L NaCl, 1 mmol/L EDTA, 0.2% Nonidet P-40, and 0.5 mmol/L DTT) at 25°C for 20 minutes. Specific competition was performed by adding 100 ng of unlabeled doublestranded oligonucleotide probe to the nuclear extract from the sample with the greatest nuclear binding, although, for nonspecific competition, 100 ng of unlabeled double-stranded mutant oligonucleotide probe that does not bind NF-κB was added. The mixture was separated by electrophoresis on a 5% polyacrylamide gel in 1 × Tris glycine EDTA buffer. Gels were vacuum dried and subjected to autoradiography. Western blot analysis. Whole cell lysates (100,000 cells/group) were prepared with 1% SDS, 150 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.4), 2 mmol/L sodium orthovanadate, 10 µg/mL leu-

peptin, 50 mmol/L NaF, 5 mmol/L EDTA, and 1 mmol/L phenylmethylsulfonyl fluoride. Lysates were diluted with 2× Laemmli buffer followed by immediate boiling for 5 minutes and then separated by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Blots were then probed with 1:1000 polyclonal rabbit anti-IκBα (Santa Cruz Laboratories). After incubation with 1:5000 goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech), blots were developed with the use of an enhanced chemiluminescence system. Equal sample loading was confirmed by Coomassieblue gel staining. Statistical analysis. Data are presented as the mean ± SE of the number of experiments indicated. When blots are shown, they are representative of 3 independent studies. Significance was assessed with the Student t test and 1-way analysis of variance with a post-hoc Tukey-Kramer multiple comparisons test. A probability value of less than .05 was considered significant. RESULTS Hypertonic modulation of macrophage cytokine production. PEMs were incubated in isotonic or HTS (500 mOsm) medium for 2 hours, washed, and resuspended in isotonic medium and then incubated in the presence or absence of LPS for 4 hours. Fig 1 shows the effect of these conditions on TNF-α and IL-10 measured in the culture supernatant by ELISA. Control cell and HTS groups did not produce any detectable TNF-α (Fig 1, A). In contrast, LPS induced a significant increase in supernatant TNF-α protein. However, HTS preconditioning inhibited LPS-induced TNF-α production by approximately 65%. Fig 1, B, shows the effect of control cell and HTS on IL-10 release. PEMs exposed to either control cell conditions or

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C Fig 4. The effect of IL-10 on the inhibition of LPS-induced PEM TNF-α production by hypertonicity. PEMs were incubated in the presence of LPS for 4 hours under isotonic conditions with or without HTS pretreatment for 2 hours. TNFα was measured in the culture supernatants by ELISA at the end of the LPS incubation period. A, Groups included unmanipulated control cells (CTRL), PEMs exposed to HTS (500 mOsm) and then resuspended in isotonic medium alone (HTS), PEMs stimulated with LPS under isotonic conditions (LPS), PEMs stimulated with LPS under isotonic conditions in the presence of anti–IL-10 neutralizing antibody (25 µg/mL: aIL-10 + LPS), cells that were preconditioned with HTS and then resuspended in isotonic medium and stimulated with LPS (HTS + LPS), and cells that were preconditioned with HTS and then resuspended in isotonic medium with anti–IL-10 neutralizing antibody (25 µg/mL) and then stimulated with LPS (HTS + aIL-10 + LPS). B, TNF-α protein was assessed as in A, with the addition of the IL-10 receptor antagonist 1b1.2. Additional groups included PEMs stimulated with LPS in the presence of the IL-10 receptor antagonist (10 µg/mL; LPS + I), and cells that were preconditioned with HTS for 2 hours and then resuspended in isotonic medium with the IL-10 receptor antagonist (10 µg/mL) and then stimulated with LPS (HTS + LPS + I). C, PEMs were harvested from mice homozygous for a deletion of the IL-10 gene and incubated in the presence of LPS.

HTS alone released little IL-10, although stimulation of macrophages with LPS caused a significant increase in IL-10 release. Preconditioning with HTS for 2 hours augmented LPS-induced IL-10 production, which resulted in a 2-fold increase in IL-10 levels compared with the isotonic LPS group. These results suggest that HTS modulates the profile of stimulated macrophage cytokine production in vitro. In addition, the augmented IL-10 release by HTS plus LPS-treated cells compared with LPS alone suggests that the inhibition of LPS-induced TNF release after HTS exposure was not due to a loss of viability of cells during the incubation.

TNF and IL-10 mRNA expression after HTS exposure. To determine whether altered PEM LPSinduced cytokine expression with hypertonic preconditioning was due to altered gene expression, Northern blot analysis was used to assess TNF and IL10 mRNA at 1 hour after LPS exposure (Fig 2). LPS exposure of isotonic pretreated cells significantly increased TNF and IL-10 mRNA expression compared with control cells. Similar to changes observed at the protein level, hypertonic preconditioning followed by LPS prevented LPS-induced TNF mRNA expression and augmented LPS-induced levels of IL10 mRNA. Interestingly, hypertonic pretreatment

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B Fig 5. NF-κB activation. Nuclear and cytoplasmic proteins were isolated from isotonic and hypertonic (500 mOsm × 2 hours) pretreated cells 5 to 60 minutes after LPS incubation under isotonic conditions. A, A representative EMSA is shown demonstrating NF-κB binding to κB consensus sequence of HIV-1 enhancer region. Treatment groups included control cells (CTRL), isotonic-LPS (LPS), hypertonic pretreatment for 2 hours followed by isotonic medium alone (HTS), and hypertonic pretreatment for 2 hours followed by LPS exposure under isotonic conditions (HTS + LPS). Cold competition with the use of an excess of unlabeled probe is also shown for the 60 minutes of LPS sample on the right. B, A representative Western blot for cytoplasmic IκBα protein.

alone increased both TNF and IL-10 mRNA levels, a change that was not mirrored in levels of protein expression. These results suggest that 1 mechanism by which HTS modulates the inflammatory response of macrophages is to alter levels of pro- and antiinflammatory cytokine gene expression. Assessment of macrophage suppressor of cytokine signaling-3 expression. The suppressor of cytokine signaling (SOCS) proteins are a recently described family of proteins known to mediate negative feedback of cytokine signaling pathways.15,16 Importantly, the ability of IL-10 to induce de novo synthesis of SOCS-3 in monocytes correlates with its ability to inhibit the expression of proinflammatory genes, which suggests that SOCS-3 may be an important mediator of IL-10 anti-inflammatory activity.15,16 Because hypertonic preconditioning augments IL-10 expression in PEMs, SOCS-3 expression was assessed by Northern blot analysis as an index of biologic activity of IL-10 in this model (Fig 3). SOCS-3 mRNA expression was assessed after cells were treated for 2 hours of 500 mOsm HTS or isotonic conditions followed by LPS exposure (1 µg/mL) under isotonic conditions for a 1or 2-hour incubation period. SOCS-3 mRNA expression was maximal in HTS + LPS and HTSalone groups. SOCS-3 mRNA expression was

detectable as early as the 1-hour time point and was further increased after a 2-hour incubation period, as compared with PEMs that did not undergo hypertonic pretreatment before LPS, which did not exhibit significant levels of SOCS-3 mRNA until 2 hours of LPS exposure had elapsed. Control cells did not exhibit SOCS-3 mRNA expression. Thus, augmented levels of IL-10 in hypertonic preconditioned cells after LPS exposure correlate with increased SOCS-3 mRNA expression, which suggests that IL-10 is biologically active in this model. Role of augmented IL-10 in hypertonicitymediated inhibition of macrophage TNF-α production in vitro. Exogenous IL-10 administration has been shown to protect against lethal endotoxemia and bacteremia and to decrease LPSinduced TNF-α production.17 Because HTS was shown to augment IL-10 expression in response to a second inflammatory stimulus (LPS) in vitro while inhibiting TNF-α production, we hypothesized that augmented IL-10 after hypertonic preconditioning might play a direct role in the inhibition of TNF-α expression. Three separate approaches were taken to determine the role of IL-10. In the first series of experiments, biologic activity of IL-10 was blocked through the addition of anti–IL-10 neutralizing antibody (25 µg/mL)

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Fig 6. Possible mechanisms that underlie hypertonic inhibition of PEM TNF-α expression. Schematic outlines possible mechanisms of hypertonic inhibitory effect.

to the culture medium immediately before LPS stimulation (Fig 4, A). Although control cells and hypertonic preconditioned cells (HTS) exhibited no detectable TNF-α production, LPS alone significantly increased macrophage TNF-α production. Similar to previous studies, hypertonic preconditioning inhibited LPS-induced TNF-α production. However, the addition of IL-10 neutralizing antibody to hypertonic preconditioned macrophages before stimulation with LPS did not result in any significant changes to LPS-induced TNF-α production as compared with HTS + LPS cells alone. The results of these initial studies that demonstrated the ability of HTS to inhibit LPSinduced TNF-α production in the presence of a neutralizing antibody against IL-10 would suggest that IL-10 does not play a direct role in the hypertonic inhibition of TNF-α. A second approach was taken to inhibit IL-10 biologic activity by treating cells with the IL-10 receptor antagonist 1B1.2 (10 µg/mL) before activation with LPS.18 As shown in Fig 4, B, IL-10 receptor blockade also failed to reverse the inhibitory effect of HTS on LPSinduced macrophage TNF-α production. These results are consistent with the concept that augmented IL-10 expression after hypertonic preconditioning of macrophages does not play a direct role in the inhibition of TNF-α production by HTS. Finally, we examined the ability of HTS to block LPS-induced TNF-α production in the complete absence of IL-10 by using macrophages harvested from mice homozygous for a targeted

deletion of the IL-10 gene (Fig 4, C). Control and HTS cells did not produce TNF-α at detectable levels. Although baseline production of TNF-α after LPS stimulation alone in IL-10–deficient cells was approximately double the level produced by wild type macrophages harvested from BALB-c mice, hypertonic preconditioning still resulted in more than a 50% inhibition of LPSinduced TNF-α production. Considered in aggregate, these studies provide strong evidence that the augmented IL-10 release in LPS-treated cells, which were pre-exposed to HTS, was not responsible for the inhibition of macrophage TNF-α production in vitro. The effect of hypertonic preconditioning on macrophage NF-κB activation. Nuclear translocation of the transcription factor NF-κB and of the binding to the promoter region of the TNF-α is an important step that leads to de novo TNF-α mRNA transcription.19 By contrast, NF-κB activation does not play a central role in the production of IL-10 by activated macrophages.20 NF-κB activation in PEMs was assessed by EMSA to determine whether the ability of hypertonic preconditioning to inhibit TNF-α expression may be due to altered activation of this proinflammatory signaling pathway (Fig 5, A). Despite the ability of HTS to inhibit TNF-α protein and gene expression after LPS treatment, it had no effect on NF-κB translocation at either 5 or 60 minutes after LPS exposure. Indeed, both HTS alone and HTS-LPS caused a small increase in NFκB compared with their controls. In support of

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these findings, degradation of I-κB after LPS (a prerequisite for NF-κB translocation) did not differ, whether cells were pretreated with HTS or isotonic saline solution (Fig 5, B). DISCUSSION Resident tissue macrophages are thought to play a prominent role in modulating the tissue injury associated with hepatic I/R and in acute respiratory distress syndrome.11,21 Data from our in vivo model of hepatic I/R that show that HTS preconditioning induces a shift in the profile of pro- and anti-inflammatory cytokines toward increased anti-inflammatory IL-10 during the macrophage-mediated phase of reperfusion injury suggest that HTS modulates the response of resident hepatic macrophages to I/R. Based on our experience with the beneficial effects of HTS in vivo in our models of posthemorrhage lung injury and hepatic I/R, we evaluated the effect of HTS on macrophages in vitro.6 Similar to the pattern of TNF-α and IL-10 expression we observed in vivo with HTS preconditioning, hypertonic exposure of murine peritoneal macrophages before LPS activation inhibited TNFα and augmented LPS-induced IL-10. These results confirm an ability of HTS to modulate the profile of pro- and anti-inflammatory cytokine expression. Others have also shown that hypertonicity is capable of inhibiting macrophage proinflammatory cytokine expression. Bode et al22 demonstrated in vitro that HTS inhibits the production of IL-6 by stimulated KC and that this effect may be due to altered mitogen activated protein kinase activation. Studies that involved hypertonic dextrose or amino acid exposure of human macrophages in the context of peritoneal dialysis have described inhibition of proinflammatory cytokine expression.23 Hypertonic inhibition of LPS-induced macrophage TNF expression could be prevented by the addition of indomethacin, which suggests a role for hypertonicity-induced prostaglandin production in the inhibitory effect.24 In a study of Kupffer cells that were stimulated with LPS under hypertonic conditions, Zhang et al25 found that prostaglandin production by Kupffer cells was increased in a dosedependent manner with increasing hypertonicity and was accompanied by a strong induction of COX-2 mRNA. The mechanisms underlying this effect are unclear. In the present studies, we show that alterations in protein expression are mirrored by changes in mRNA expression. We investigated the possibility that reduced TNF mRNA levels may be related to reduced signaling through NF-κB, because this pathway is known to contribute to TNF-α mRNA transcription (Fig 6). Despite reduc-

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ing TNF mRNA steady state levels, HTS had no effect on LPS-induced NF-κB translocation. This suggests either that reduced transcription may have occurred through an NF-κB independent pathway or alternatively that reduced TNF-α mRNA steady-state levels were related to the altered stability of the mRNA. In this regard, our results that demonstrated a relatively greater inhibition of TNF-α protein as compared with the level of TNF-α mRNA inhibition by hypertonic preconditioning suggest that posttranslational effects may also be important. Hypertonicity was previously shown to inhibit adenosine triphosphate–induced posttranslational processing of the immature form of IL-1β in monocytes.26 Further studies will help discern these possibilities. The potent anti-inflammatory cytokine, IL-10, is capable of inhibiting the synthesis of proinflammatory cytokines (such as TNF) and adhesion molecules (such as ICAM-1).27,28 Given its known inhibitory effects, we hypothesized that IL-10 might contribute to inhibition TNF release after LPS. The fact that IL-10 had biologic activity in these cells was supported by its enhanced upregulation of SOCS-3. Despite this, studies that used IL-10 neutralizing antibodies, IL-10 receptor blockade, and macrophages harvested from IL-10–deficient mice did not support a direct role for IL-10 in the inhibition of LPS-induced macrophage TNF-α production in vitro. Other mechanisms are clearly responsible and are the subject of present investigation. In conclusion, pretreatment with HTS was associated with an altered cytokine profile after LPS, in which TNF-α expression was inhibited and expression of IL-10 was augmented. The ability of HTS to shift the balance of cytokine production by activated macrophages toward augmented counterinflammatory cytokine production is consistent with the notion that HTS might modulate the activation of macrophages after inflammatory stimuli in vivo and thus prevent the development of organ injury. REFERENCES 1. Vassar MJ, Fischer RP, O’Brien PE, Bachulis BL, Chambers JA, Hoyt DB, et al. A multicenter trial for resuscitation of injured patients with 7.5% sodium chloride: the effect of added dextran 70: the multicenter group for the study of hypertonic saline in trauma patients. Arch Surg 1993;128:1003-11. 2. Holcroft JW, Vassar MJ, Perry CA, Gannaway WL, Kramer GC. Use of a 7.5% NaCl/6% dextran 70 solution in the resuscitation of injured patients in the emergency room. Prog Clin Biol Res 1989;299:331-8. 3. Mattox KL, Maningas PA, Moore EE, Mateer JR, Marx JA, Aprahamian C, et al. Prehospital hypertonic saline/dextran infusion for post-traumatic hypotension: the USA Multicenter Trial. Ann Surg 1991;213:482-91.

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Surgery Volume 130, Number 2 4. Wade CE, Kramer GC, Grady JJ, Fabian TC, Younes RN. Efficacy of hypertonic 7.5% saline and 6% dextran-70 in treating trauma: a meta-analysis of controlled clinical studies. Surgery 1997;122:609-16. 5. Simma B, Burger R, Falk M, Sacher P, Fanconi SA. Prospective, randomized, and controlled study of fluid management in children with severe head injury: lactated Ringer’s solution versus hypertonic saline [see comments]. Crit Care Med 1998;26:1265-70. 6. Rizoli SB, Kapus A, Fan J, Li YH, Marshall JC, Rotstein OD. Immunomodulatory effects of hypertonic resuscitation on the development of lung inflammation following hemorrhagic shock. J Immunol 1998;161:6288-96. 7. Angle N, Hoyt DB, Cabello-Passini R, Herdon-Remelius C, Loomis W, Junger WG. Hypertonic saline resuscitation reduces neutrophil margination by suppressing neutrophil L selectin expression. J Trauma 1998;45:7-12. 8. Rizoli SB, Rotstein OD, Kapus A. Cell volume-dependent regulation of L-selectin shedding in neutrophils: a role for P38 mitogen-activated protein kinase. J Biol Chem 1999;274:22072-80. 9. Rizoli SB, Kapus A, Parodo J, Fan J, Rotstein OD. Hypertonic immunomodulation is reversible and accompanied by changes in CD11b expression. J Surg Res 1999;83:130-5. 10. Oreopoulos GD, Hamilton J, Rizoli SB, Fan J, Lu Z, Li YH, et al. In vivo and in vitro modulation of intercellular adhesion molecule (ICAM)-1 expression by hypertonicity [in process citation]. Shock 2000;14:409-14. 11. Jaeschke H. Mechanisms of reperfusion injury after warm ischemia of the liver. J Hepatobiliary Pancreat Surg 1998;5:402-8. 12. Mawet E, Shiratori Y, Hikiba Y, Takada H, Yoshida H, Okano K, et al. Cytokine-induced neutrophil chemoattractant release from hepatocytes is modulated by Kupffer cells. Hepatology 1996;23:353-8. 13. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. 14. Deryckere F, Gannon F. A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 1994;16:405. 15. Donnelly RP, Dickensheets H, Finbloom DS. The interleukin-10 signal transduction pathway and regulation of gene expression in mononuclear phagocytes. J Interferon Cytokine Res 1999;19:563-73. 16. Stoiber D, Kovarik P, Cohney S, Johnston JA, Steinlein P, Decker T. Lipopolysaccharide induces in macrophages the

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