Introduction

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Lipid peroxidation is an autocatalytic process initiated by reactive oxygen species that are generated under conditions of cellular oxidative stress (Esterbauer et al., 1991). Peroxidation of cellular polyunsaturated fatty acids results in the formation of chemically reactive lipid aldehydes capable of diffusing from their site of origin (Benedetti et al., 1979). A major end product produced during lipid peroxidation is the ,-unsaturated aldehyde, 4-hydroxy-trans-2-nonenal (4-HNE), believed to be largely responsible for many of the cytopathologic effects observed during oxidative stress (Poli and Schaur, 2000). 4-HNE exhibits a wide range of biological activities including inhibition of RNA and DNA synthesis, stimulation of neutrophil migration (Esterbauer et al., 1991), enzyme inhibition (Uchida and Stadtman, 1993), and activation of stress-signaling pathways via transcription factors (Camandola et al., 1997; Parola et al., 1998) and kinase pathways (Uchida et al., 1999), as well as inhibition of the NF-B signaling pathway (Page et al., 1999, Ji et al., 2001). Many of the effects of 4-HNE have been attributed to the ability of this compound to chemically modify cellular macromolecules because this ,-unsaturated aldehyde reacts rapidly with nucleophilic functional groups of proteins and DNA (Esterbauer et al., 1991).

Kupffer cells are macrophages normally present in the liver and primarily involved in removal of particulate and foreign materials from the portal circulation (Laskin, 1990). During exposure to various hepatotoxins, Kupffer cells release biologically active mediators, including cytokines and prostaglandins, that appear to be important in inflammation or function in regeneration of the liver (Decker, 1990). For instance, interleukin-6 (IL-6) has been demonstrated to be essential in liver regeneration after partial hepatectomy and by attenuating carbon tetrachloride (CCl₄)-induced acute and chronic liver injury and fibrosis (Streetz et al., 2000). Based on the potential of 4-HNE to interact with various cellular pathways including the NF-B system, we hypothesized that this aldehyde could also affect IL-6 cytokine production by modulating the activity of the NF-B system in primary rat Kupffer cells.

The data presented here demonstrate that 4-HNE decreases IL-6 production in rat Kupffer cells by inhibiting NF-B activation through mechanisms involving decreased phosphorylation of IB and stabilization of IB levels. The results of this study are novel in that they are the first to demonstrate potential functional implications of 4-HNE treatment in primary rat Kupffer cells through modulation of IL-6 production.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) and were analytical grade or better. All solutions were prepared in deionized and distilled water.

Animals. Male high alcohol sensitivity and low alcohol sensitivity rats routinely weighing between 250 and 350 g were obtained from the University of Colorado Alcohol Research Center. These animals have been selected for genotypically based central nervous system sensitivities after acute ethanol administration that are independent of the hepatic enzymatic alcohol or aldehyde detoxification pathways (Draski et al., 1992). Therefore, the phenotypic responses of Kupffer cells from these animals to 4-HNE are predictably similar to those of other genetic stocks of rats. The animals received humane care, and all experimental protocols were reviewed, consistent with National Institutes of Health guidelines, and were approved by the Institutional Animal Care and Use Committee at the University of Colorado Health Sciences Center.

Kupffer Cell Isolation. Kupffer cells were isolated according to the well characterized procedure outlined elsewhere (Petroft and Smersrod, 1987). Briefly, the liver was perfused through the portal vein with 400 ml of Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution (HBSS) at 37°C at a flow rate of 25 ml/min. Subsequent perfusion with complete HBSS containing 0.025% collagenase IV and 0.01% DNase at 37°C occurred for 5 min. After complete digestion, the liver was removed and the cells were dispersed in collagenase buffer. Parenchymal cells were pelleted by centrifugation at 50g for 3 min. Additional centrifugations at 50g removed any remaining hepatocytes from the nonparenchymal fraction. The 50g supernatant containing the nonparenchymal cells was washed twice with HBSS to remove dead cells and other cell debris. Nonparenchymal cells were then centrifuged on a Percoll density cushion at 1000g for 15 min, and the Kupffer cell fraction was collected and washed again. Cells were seeded onto tissue culture plates at a density of 5 × 10⁶/ml and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate at 37°C with 5% CO₂. Nonadherent cells were removed after 0.5 h by washing the plated cells and replacing the culture medium. Adherent cells were analyzed for their ability to phagocytose latex beads, indicating that they are viable Kupffer cells.

Measurement of Cytokine Release in Culture Medium. IL-6 cytokine production by Kupffer cells was determined after different 4-HNE treatments. Initial studies involved treating Kupffer cells with 4-HNE (0.1-10 µM) for 1 h, followed by LPS (500 ng/ml; from Escherichia coli, serotype 055:B5) administration for an additional 8 h. Similarly, Kupffer cells were first activated with LPS (500 ng/ml) for 1 h followed by administration of 4-HNE (0.1-10 µM) for 8 h. Previous studies from this laboratory have determined that the 4-HNE IC₅₀ value for isolated Kupffer cells treated in the above manner is 32 µM (Luckey and Petersen, 2001). Therefore, the concentrations of 4-HNE used in the present studies are substantially below those considered to be toxic or growth-inhibitory. Aliquots of the medium from both treatments were obtained and kept at 80°C until assayed. IL-6 in the culture medium was measured using an enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems, Minneapolis, MN).

Development of Specific Oligonucleotide Probe Sets for bDNA Analysis. The bDNA signal amplification assay is a nonpolymerase chain reaction- and nonradioactive-based method of RNA analysis based upon the well established enzyme-linked immunosorbent assay utilizing a multinucleotide approach (Hartley and Klaassen, 2000). The gene sequence of IL-6 and IB (accession nos. A34247 and Q63746) was accessed from GenBank and developed as previously described (Hartley and Klaassen, 2000). For oligonucleotide probe development, the nucleotide sequences were aligned using CLUSTALW with software provided by OMIGA (Oxford Molecular Group, Inc., Oxford, UK) to identify specific target regions unique to the IL-6 or IB gene. The target sequences were determined by ProbeDesigner Software Version 1.0 (Bayer Diagnostics, East Walpole, MA), and multiple and specific probes were developed to the mRNA transcript. All oligonucleotide probes were developed with a melting temperature (T_m) of approximately 63°C that enables hybridization conditions to be held constant at 53°C. Each probe was analyzed for nucleotide comparison with the National Center for Biotechnological Information to ensure minimal cross-reactivity with other rat gene sequences. Oligonucleotides were synthesized by Operon Technologies (Palo Alto, CA) and obtained in desalted and lyophilized preparations. All probes including blocker probes, capture extenders, and label extenders were diluted in 1.0 ml of 10 mM Tris-HCl, pH 8.0, with 1 mM EDTA and stored at 20°C.

bDNA Assay. Kupffer cells were treated as previously described for IL-6 protein assay. Total RNA was analyzed using the Quantigene bDNA Signal Amplification Kit (Bayer Diagnostics) according to the manufacturer's protocol. Briefly, specific oligonucleotide probe sets including blocker probes, capture probes, and label probes were combined and diluted to 50 fmol/µl in the lysis buffer supplied in the Quantigene bDNA Signal Amplification Kit. All reagents including lysis buffer, capture hybridization buffer, amplifier/label buffer, wash A and D, and substrate solution were supplied in the Quantigene bDNA Signal Amplification Kit. Total cellular mRNA (50 µg) was added to each well of a 96-well plate containing capture hybridization buffer and 100 µl of each probe set containing all probes for a given transcript (blocker probes, capture probes, and label probes). Hybridization of total RNA to the specific probe set occurred at 53°C overnight. The plate was then removed from the incubator, cooled to room temperature, and subsequently rinsed with wash A. A solution of bDNA amplifier molecules (50 µl/well) diluted in amplifier/label probe buffer was allowed to hybridize to the samples for 30 min at 53°C. Again, the plate was cooled to room temperature and washed with wash A. Sample complexes of bDNA-RNA were then hybridized with a solution containing the label probe (50 µl/well) diluted in amplifier/label probe buffer for 15 min at 53°C. The plate was removed and cooled to room temperature, and each well was washed twice with wash A and three times with wash D. Alkaline-phosphate-mediated luminescence was initiated by the addition of the dioxetane substrate solution (50 µl/well). The enzymatic reaction occurred for 30 min at 37°C, and the luminescence of each sample was measured. Values for the relative luminescence were expressed as the ratio of expression for the specific gene of interest (IL-6 or IB) to glyceraldehyde-3-phosphate dehydrogenase. The levels of glyceraldehyde-3-phosphate dehydrogenase were unaffected by 4-HNE treatment in this study; therefore, mRNA levels were normalized to this gene.

Adenoviral Infection. Adenovirus expressing the IB S32A/S36A super-repressor was originally constructed in the laboratory of David Brenner, University of North Carolina at Chapel Hill (Jobin et al., 1998). Kupffer cells were isolated and plated at 5 × 10⁶/ml in six-well plates overnight. The following day, the cells were infected with Ad5IB or Ad5GFP in serum-free medium at a multiplicity of infection of 10:1 (adenoviral particulates to Kupffer cells) for 6 h. Nonadherent adenovirus was removed by washing with phosphate-buffered saline, and cells were cultured overnight in fresh medium-containing serum. Cells were treated with LPS (1 µg/ml) for 6 h, at which time medium was removed and analyzed for the presence of IL-6 protein as previously described.

NF-B Assay. NF-B was analyzed by a sensitive multiwell colorimetric assay for active NF-B as previously reported (Renard et al., 2001). Kupffer cells were preincubated with 4-HNE for 1 h followed by LPS (500 ng/ml) administration for 1 h. Cells were rinsed with cold HBSS, and 100 µl of lysis buffer (20 mM HEPES, pH 7.5, 0.35 M NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl₂·6H₂O, 0.5 mM EDTA, 0.1 mM EGTA) containing a protease inhibitor cocktail was applied to the cells. The cells were then scraped and placed on ice for 10 min. The lysate was centrifuged for 10 min at 14,000 rpm. The supernatant constituting the total cellular extract was frozen at 70°C. The cellular extract was added to the microplate wells at a protein concentration of 1 µg of cell extract per well. The wells were precoated with the NF-B consensus oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3'. Incubation of whole-cell lysate occurred in a binding buffer supplied by the manufacturer. After a 1-h incubation at room temperature with mild agitation, microwells were washed three times with PBS + 0.1% Tween 20. Rabbit anti-NF-B antibodies to p65 (1:2,000) were incubated in each well at room temperature for 1 h. Each well was washed, and a peroxidase-conjugated goat anti-rabbit IgG antibody (1:2,000) was incubated in each well for 1 h at room temperature. The microwells were washed, and tetramethylbenzidine was incubated in each well for 5 min, at which time 100 µl of stop solution was added to each well, after which the optical density was determined at 405 nm. The results are expressed after subtraction of the blank values. The specificity of binding was examined by competition with both a 50-fold excess of unbound oligonucleotide and a 50-fold excess of an oligonucleotide with three bases mutated (5'- AGTTGAGCTCACTTTCCCAGGC-3'), which cannot bind active NF-B.

Immunoprecipitation. To determine IB and p65 protein interactions, cells were lysed in an immunoprecipitation buffer (IP) consisting of 150 mM NaCl, 50 mM Tris, and 1% Nonidet P-40, pH 8.0, and a mammalian cell-specific protease inhibitor cocktail (Sigma-Aldrich). Total cellular extracts (100 µg of protein) were incubated with anti-p65 antibody (1 µg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA) in IP buffer for 3 h at room temperature on a rocker. The antibody-protein mixture was agitated at 1400 rpm with Protein A/G agarose (Santa Cruz Biotechnology) overnight at 4°C. The immunoprecipitates were washed four times with IP buffer. The washed immunoprecipitates were incubated in 30 µl of 1× electrophoresis buffer and heated at 100°C for 5 min. The beads were spun out and the supernatant was resolved by SDS-PAGE, and the levels of p65/IB interactions were analyzed by Western blot.

Western Blotting. Cellular supernatant (15 µg) was mixed with 1 volume of sample loading buffer and was heated to 100°C for 2 min prior to SDS-PAGE using a 4% stacking and a 12% resolving gel, run at 2 h at 50 mA per gel. Fractionated proteins were electrophoretically transferred to polyvinylidene difluoride microporous membranes in Towbin's buffer (192 mM glycine, 25 mM Tris-HCl, and 20% methanol). After 1 h of transferring at 43 mA, blots were blocked by shaking overnight in 5% nonfat dry milk in TBST buffer (25 mM Tris, pH 7.6, 146 mM NaCl, 0.1% Tween 20) at 4°C. The blotted polyvinylidene difluoride membranes were incubated for 2 h at room temperature (RT) with anti-IB (Cell Signaling Technology Inc., Beverly, MA) or anti-phospho-specific IB antibodies (Cell Signaling Technology Inc.) in TBST buffer with 5% nonfat milk. The blots were subsequently washed four times for 15 min at RT. Furthermore, the immunoblots were incubated for 1 h at RT with a 1:2500 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies in TBST buffer with 5% nonfat milk. Once again, the immunoblots were washed, and the blotted protein bands were detected by chemiluminescence using an enhanced chemiluminescence Western blotting kit.

Statistical Analysis. Statistical analyses were performed using GraphPad Prism, version 3.0 (GraphPad Software Inc., San Diego, CA). The mean ± S.E.M. was determined for each treatment group in three to eight independent experiments. Statistical analysis was performed by Student's t test, and differences between treatment groups were designated significant at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

4-HNE Suppresses IL-6 Production by Rat Kupffer Cells. To determine whether 4-HNE is capable of modulating cytokine production, Kupffer cells were incubated with low micromolar concentrations of 4-HNE (0.1-10 µM) for 1 h, followed by stimulation with LPS (500 ng/ml) for 1 h. The medium was analyzed by an ELISA revealing that incubation of Kupffer cells with LPS significantly induced increases in the production of IL-6 protein (Fig. 1A). Whereas IL-6 was reduced approximately 10% in Kupffer cells exposed to 0.1 µM 4-HNE, the decrease was not statistically significant. However, IL-6 production was reduced to approximately 50%, 75%, and 90% compared with LPS-treated Kupffer cells after treatment with 1, 5, and 10 µM 4-HNE, respectively. Similarly, when Kupffer cells were stimulated with LPS for an hour and subsequently incubated with the same concentrations of 4-HNE, a similar pattern of reduced IL-6 production was evident (Fig. 1B). Interestingly, LPS-induced IL-6 protein production in Kupffer cells was also examined after exposure to low-micromolar concentrations of another ,-unsaturated aldehyde, trans-2-hexenal, which also markedly decreased IL-6 release (data not shown). These data suggest that the electrophilic ,-unsaturated aldehydes in general and 4-HNE specifically are potent inhibitors of IL-6 production. Consequently, these results indicate that 4-HNE modulates IL-6 production in Kupffer cells not only before, but also after LPS-mediated activation.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   4-HNE inhibits LPS-induced IL-6 protein production in Kupffer cells. A, Kupffer cells were treated with 4-HNE (0.1-10 µM) for 1 h and subsequently stimulated for 8 h with LPS (500 ng/ml). B, Kupffer cells were stimulated with LPS (500 ng/ml) for 1 h and subsequently treated with 4-HNE (0.1-10 µM) for 8 h. IL-6 protein concentrations in the cellular supernatant were evaluated by an ELISA method as indicated under Materials and Methods. Statistical significance was determined by Student's t test (*, significantly different from LPS stimulation alone, P < 0.05). Values represent mean ± standard error for n = 5 to 8 experiments.

4-HNE Decreases IL-6 mRNA in Rat Kupffer Cells. It was important to establish whether 4-HNE-mediated decreases in IL-6 protein production occur concurrently with a reduction in IL-6 mRNA. As demonstrated by the bDNA signal amplification assay, and consistent with the data presented in Fig. 1, A and B, the IL-6 message was decreased to below LPS-stimulated levels in response to treatment with concentrations of 4-HNE ranging from 1 to 10 µM (Fig. 2A). Maximal reduction in gene expression of IL-6 was observed at 5 µM 4-HNE, representing an approximately 70% decrease compared with LPS-stimulated Kupffer cells not exposed to 4-HNE. As previously described in the experiments analyzing IL-6 protein levels, 4-HNE also affected IL-6 gene expression following Kupffer cell activation by LPS for an hour (Fig. 2B). Collectively, these data suggest that 4-HNE decreases protein production of IL-6 in Kupffer cells accompanied with depressed IL-6 mRNA levels. Additional studies have determined that similar concentrations of 4-HNE also attenuate LPS-mediated tumor necrosis factor- mRNA and protein production in Kupffer cells (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   4-HNE decreases LPS-induced IL-6 mRNA levels in Kupffer cells. A, Kupffer cells were treated with 4-HNE (0.1-10 µM) for 1 h and subsequently stimulated for 8 h with LPS (500 ng/ml). B, Kupffer cells were stimulated with LPS (500 ng/ml) for 1 h and subsequently treated with 4-HNE (0.1-10 µM) for 8 h. Cells were immediately lysed, and IL-6 gene expression was analyzed by the bDNA assay described under Materials and Methods. Statistical significance was determined by Student's t test (*, significantly different from LPS stimulation alone, P < 0.05). Values represent mean ± standard error for n = 5 to 8 experiments.

NF-B Modulates IL-6 Production in Rat Kupffer Cells. Since NF-B is a key transcription factor in regulation of the IL-6 gene in certain cell lines (Libermann and Baltimore, 1990), the associated role of this transcription factor and IL-6 production in rat Kupffer cells was evaluated. Activation of NF-B requires both phosphorylation and degradation of its inhibitory protein, IB (Brown et al., 1995). Therefore, to block NF-B activity, the IB S32A/S36A mutant, resistant to phosphorylation and degradation, was delivered to Kupffer cells using a recombinant adenoviral vector (Ad5IB). This super-repressor has been previously demonstrated to inhibit NF-B activity and cytokine production (tumor necrosis factor- and IL-8) in human intestinal epithelial cells (Jobin et al., 1998) and rat Kupffer cells (Wheeler et al., 2001). LPS-induced IL-6 protein production was suppressed by approximately 70% in Ad5IB-infected Kupffer cells (Fig. 3). Only minor changes in LPS-stimulated IL-6 protein levels were observed with Ad5GFP-infected Kupffer cells (Fig. 3), and these cells did not generate IL-6 protein when either adenovirus was administered alone (data not shown). These results indicate that NF-B activity is central in the transcriptional regulation, expression, and production of IL-6 in rat Kupffer cells.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Inhibition of LPS-induced IL-6 concentrations in Ad5IB-infected Kupffer cells. Primary Kupffer cells were infected with either Ad5IB or Ad5GFP for 8 h, after which cells were stimulated with LPS (1 mg/ml) for 6 h. IL-6 concentrations were measured from cell supernatants using an ELISA method. Statistical significance was determined by Student's t test (*, significantly different from LPS stimulation alone, P < 0.05). Values represent mean ± standard error for n = 3 to 4 experiments.

4-HNE Blocks NF-B Binding in Rat Kupffer Cells. To correlate 4-HNE effects with IL-6 gene expression and protein production, NF-B DNA binding was evaluated. NF-B binding to its consensus sequence was determined according to a validated and sensitive ELISA in which the activated transcription factor is captured by an immobilized probe (Renard et al., 2001). As evident in Fig. 4, LPS stimulation results in activation of NF-B binding activity. Pretreatment of Kupffer cells with 4-HNE (0-10 µM) for 60 min resulted in a significant, concentration-dependent decrease in NF-B binding, with 4-HNE concentrations of 5 µM reducing NF-B binding to its consensus sequence by 50%. Specificity of the nuclear binding activity of NF-B was confirmed by competing the bound capture probe with excess (50-fold) wild-type NF-B oligonucleotide probe (Fig. 4), whereas competition with excess (50-fold) mutated oligonucleotide showed little reduction in NF-B binding. These data provide evidence that 4-HNE reduces IL-6 production at the transcriptional level by decreasing NF-B binding.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   4-HNE blocks NF-B transcriptional activity in Kupffer cells. Kupffer cells were treated with 4-HNE (0.1-10 µM) for 1 h and subsequently stimulated with LPS (500 ng/ml) for 1 h. NF-B activity was analyzed by a colorimetric assay as previously described (Renard et al., 2001). The figure indicates percentage of NF-B activity normalized to the 100% LPS control. The specificity of binding was evaluated by adding a 50-fold excess of either the wild-type or the mutated NF-B-binding consensus sequence. Statistical significance was determined by Student's t test (*, significantly different from LPS stimulation alone, P < 0.05). Values represent mean ± standard error for n = 5 to 8 experiments.

4-HNE Stabilizes IB and Decreases IB Phosphorylation in Rat Kupffer Cells. As previously discussed, upon stimulation by specific activators of NF-B, IB is phosphorylated and degraded, thereby allowing translocation of free NF-B to the nucleus. Consequently, IB and phosphorylated IB levels in Kupffer cells were analyzed by Western blot analyses. The immunoblot in Fig. 5A presents the time course of IB and phosphorylated IB in Kupffer cells treated with LPS (500 ng/ml). No detectable changes in IB were evident upon stimulation of Kupffer cells with LPS (Fig. 5A), consistent with reports of a high synthetic rate for new IB protein (Rice and Ernst, 1993). LPS did initiate increases in phosphorylated IB levels in Kupffer cells following 20 min of treatment (Fig. 5A), and a reduction of phosphorylated IB to levels comparable with those of controls was observed by 120 min. To determine a possible mechanism of suppressed NF-B activation by 4-HNE, Kupffer cell extracts were also analyzed for IB and phosphorylated IB (Fig. 5B). These results demonstrate that 4-HNE stabilizes IB protein in rat Kupffer cells when the aldehyde is added an hour before LPS stimulation, as evidenced by increases in IB at concentrations as low as 0.1 µM 4-HNE. Densitometric analysis of fold-induction of IB from cells treated with 4-HNE over LPS stimulation alone (0 µM 4-HNE) was 1.3 for 0.1 µM, 1.8 for 1 µM, 2.0 for 5 µM, and 2.1 for 10 µM 4-HNE (Fig. 5C). Treatment of rat Kupffer cells with 4-HNE also inhibited phosphorylation of IB with a significant, 20% decrease observed after incubation with 5 µM 4-HNE and nearly completely inhibited LPS-induced phosphorylation of IB at 10 µM 4-HNE (Fig. 5, B and D). These results suggest that increased stabilization of 4-HNE-mediated stabilization of IB is a consequence of decreased IB phosphorylation.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   4-HNE prevents IB degradation by blocking IB-phosphorylation in Kupffer cells. A, Kupffer cells were treated with LPS (500 ng/ml) for various times (0-120 min), and cellular extracts (15 µg of protein/lane) were analyzed for the levels of IB and phosphorylated IB protein by Western blot. B, additionally, Kupffer cells were treated with 4-HNE (0.1-10 µM) for 1 h and subsequently stimulated with LPS (500 ng/ml) for 1 h. IB cellular levels were analyzed and phosphorylated IB was detected by a phospho-specific antibody recognizing IB phosphorylated on Ser-32 and/or Ser-36. C and D, arbitrary densitometric analyses of three independent immunoblots for IB (C) and IB-PO4 (D). Values represent mean ± standard error.

4-HNE Affects NF-B p65 and IB Interactions. To determine whether the observable suppression of NF-B binding activity is a direct result of IB stabilization, immunoprecipitation-immunoblot analyses were conducted to evaluate interactions of IB with the p65 subunit of NF-B. The interaction between p65 and IB proteins is apparent by the presence of the immunoprecipitated, immunopositive band at approximately 41 kDa, which corresponds to the apparent molecular weight of IB (Fig. 6A). When evaluated prior to LPS stimulation, the p65 and IB complex was retained at concentrations of 4-HNE up to 5 µM. Stabilization at higher concentrations of 4-HNE was not observed. As measured by densitometry, a 50% reduction in the apparent interaction between the p65 and IB proteins was observed in Kupffer cells exposed to 10 µM 4-HNE (Fig. 6B). These results indicate that p65 and IB retain a physical interaction in the presence of 4-HNE concentrations up to 5 µM; however, the diminished interaction at higher concentrations of 4-HNE may not account for the observed decreases in NF-B binding.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   4-HNE affects the interactions of IB and p65 in Kupffer cells. A, Kupffer cells were treated with 4-HNE (0.1-10 µM) for 1 h and subsequently stimulated with LPS (500 ng/ml) for 1 h. Cellular extracts were lysed with IP buffer and immunoprecipitated with anti-p65 antibody. The effect of 4-HNE on p65/IB protein complex was analyzed by SDS-PAGE and Western blot. The immunoblots were visualized with antiserum to IB. B, arbitrary densitometric analyses of at least three independent immunoprecipitation-immunoblots. Values represent mean ± standard error.

4-HNE Decreases IB Gene Expression in Rat Kupffer Cells. To evaluate whether the effects on NF-B binding by 4-HNE are related to increases in newly synthesized IB mRNA levels, IB gene expression was analyzed. The data in Fig. 7 indicate that IB mRNA levels are suppressed by 4-HNE treatment in Kupffer cells as measured by the bDNA signal amplification assay. 4-HNE strongly inhibited IB gene expression in Kupffer cells, with a 50% reduction in gene expression at 1 µM 4-HNE and maximal inhibition occurring by administration of 5 µM 4-HNE. These results suggest that synthesis of new IB mRNA levels is suppressed by 4-HNE and is not related to the decreases in NF-B binding activity.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   4-HNE decreases LPS-mediated IB gene expression in Kupffer cells. Kupffer cells were treated with 4-HNE (0.1-10 µM) for 1 h and subsequently stimulated with LPS (500 ng/ml) for 1 h. Cells were immediately lysed and mRNA levels of IB were evaluated by the bDNA assay. Statistical significance was determined by Student's t test (*, significantly different from LPS stimulation alone, P < 0.05). Values represent mean ± standard error for n = 3 to 4 experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Novel data presented here demonstrate that treatment of rat Kupffer cells with 4-HNE decreases IL-6 gene expression and protein production by preventing activation of the NF-B signaling pathway. In addition, the results also suggest that modulation of the NF-B signaling pathway results from 4-HNE-mediated increases in cellular levels of IB and decreased phosphorylation of IB. Furthermore, these data also indicate the complexity of the interactions between the NF-B system and the cellular effects of 4-HNE.

The studies described in the present communication using physiologically relevant and nontoxic concentrations of 4-HNE (0.1-10 µM) are consistent with previous reports that relatively high concentrations of 4-HNE (25-60 µM) alter the NF-B signaling system by inhibiting IB phosphorylation in both human monocytic cells (Page et al., 1999) and colorectal cells, as well as human lung carcinoma cells (Ji et al., 2001). Both of these investigations reported that 4-HNE inhibits IB phosphorylation by inhibiting the kinases (IKK) involved in the phosphorylation of IB. Furthermore, Ji et al. (2001) suggested that the effects of 4-HNE were at the level of IKK, because unpublished data from their laboratory (C. Ji, K. R. Kozak, and L. J. Marnett, unpublished data) indicated that 4-HNE did not inhibit upstream kinases involved in regulating IKK activity, including ERK1, ERK2, JNK1, and JNK2-1 and -2.

In the present study, the effect of 4-HNE on IB in Kupffer cells was observed at 0.1 µM 4-HNE; however, this aldehyde affected phosphorylation of IB at concentrations of 5 and 10 µM 4-HNE. Additionally, there was no associated increase in IB-p65 complex corresponding with the increases in IB after 4-HNE treatment. The interaction between these proteins also decreased at 10 µM 4-HNE (Fig. 6), consequently suggesting that 4-HNE disrupts NF-B binding activity at a level independent of the kinase activity. One possible explanation for these results may be related to a previous study reporting that another ,-unsaturated aldehyde, acrolein, reduced NF-B binding to its consensus sequence through an IB-independent mechanism (Horton et al., 1999). These investigators hypothesized that acrolein interfered with the activity of NF-B by either direct inactivation of NF-B, possibly through covalent modification, or by scavenging nuclear reducing equivalents, such as glutathione, required for NF-B binding, thereby lowering the binding affinity of NF-B for DNA. Based on the fact that 4-HNE is also an ,-unsaturated aldehyde, and that 4-HNE rapidly reduces cellular glutathione levels in Kupffer cells (Luckey and Petersen, 2001), it is possible that 4-HNE may also alter NF-B activation in rat Kupffer cells by either of these mechanisms.

Recent findings indicate that the regulation of IL-6 gene expression is mediated by several transcription factors (Isshiki et al., 1990). Analysis of the IL-6 gene promoter indicates the presence of not only an NF-B binding site, but also a glucocorticoid-responsive element, an activator protein-1 binding site, and a c-fos serum-responsive element homology (Hirano et al., 1990). Furthermore, an NF-IL6-binding element having high homology with CCAAT/enhancer-binding protein has been reported to be important for IL-6 expression (Akira et al., 1990). Thus, it is possible that multiple interactions among these transcription factors may be required for the expression of IL-6. Based on the present study, it is clear that NF-B is required for the production of IL-6, but the specific 4-HNE-mediated alterations in the cross-talk among the transcription factors involved in IL-6 expression in rat Kupffer cells remains to be elucidated.

The reported potential of 4-HNE to inhibit the ubiquination/proteasome system (Okada et al., 1999) may be responsible for the observed decreases in the DNA binding of NF-B illustrated in Fig. 4. These investigators (Okada et al., 1999) proposed that 4-HNE-mediated inhibition of ubiquination/proteasome-dependent proteolysis might be partially attributed to the direct attachment of 4-HNE to the proteasome system. Relevant to the present investigation, decreased proteasome activity affects NF-B functional activity since this transcription factor is regulated at several levels by the ubiquitin-proteasome system (Palombella et al., 1994). NF-B activity is regulated not only by the proteasome system at the level of IB degradation, but also in the processing of p105 to generate the active p65-p50 NF-kb heterodimer. However, the degree to which either of these systems is affected by 4-HNE in rat Kupffer cells has not been established.

Finally, the ability of 4-HNE to form covalent modifications to protein functional groups may also play a role in the ability of this ,-unsaturated aldehyde to decrease IL-6 production in rat Kupffer cells. In addition to modifications of the ubiquination/proteasome system, 4-HNE may also form covalent adducts with either IB or NF-B or both, thereby reducing the functional cellular roles of these proteins. As previously discussed, 4-HNE adducts with NF-B may decrease DNA binding ability of this transcription factor. Not only may covalent modifications with IB result in stabilization of this protein, but 4-HNE-modified IB may also account for decreases in the corresponding IB-p65 complex at 10 µM 4-HNE as observed in Fig. 6.

Pertinent to the present study are numerous reports establishing that IL-6 is important in liver regeneration and involved both in the early stages of liver disease and through the more advanced stages of liver damage and fibrosis (Streetz et al., 2000). Therefore the functional implications of this study are very relevant. In IL-6 knockout mice, partial hepatectomy results in impaired proliferative responses during liver regeneration and ultimately in liver failure (Cressman et al., 1996). It is thought that IL-6 may be involved in triggering the G₀/G₁ phase transition of hepatocytes after hepatectomy. Additional investigations of the role of IL-6 in CCl₄-induced liver injury and hepatic fibrosis have established that during acute CCl₄-mediated liver injury, STAT3 and NF-B levels are reduced and DNA synthesis is impaired in IL-6-deficient mice compared with the IL-6 +/+ mice (Kovalich et al., 2000). These investigators also reported that following chronic CCl₄ treatment, IL-6 / mice had increased fibrosis and an elevated number of activated stellate cells compared with CCl₄-treated IL-6 wild-type mice. Similarly, studies of biliary cirrhosis induced by bile duct ligation revealed that IL-6-deficient mice had elevated serum biliary levels and developed advanced stages of biliary fibrosis more rapidly than control IL-6 +/+ mice (Ezure et al., 2000). In each of the previously cited studies, liver injury was ameliorated with exogenous treatment of the IL-6 knockout mice with supplemental IL-6 protein. Consequently, the effects of 4-HNE on IL-6 production observed in the present study are important in regard to chemically mediated liver injury, because decreased production of IL-6 by activated Kupffer cells may potentially affect liver regeneration and/or liver fibrosis during elevated oxidative stress. Furthermore, the results presented in this study also provide potential explanation to a recent investigation revealing impaired IL-6 gene expression and protein production by Kupffer cells during chronic iron overload (Olynyk and Clarke, 2001). Since the pathology of liver toxicity during iron overload involves the generation of aldehydic products (Valerio and Petersen, 1998), the production of this ,-unsaturated aldehyde may account for the reduced gene expression of IL-6 in Kupffer cells following iron overload.

The present studies have relevant functional implications in that 4-HNE prevents Kupffer cells from producing IL-6, a cytokine documented to play an important role in the proliferative and regenerative response following acute and chronic liver injury. The data also provides mechanistic details of 4-HNE-induced suppression of NF-B activity not only at the level of IKK, but also possibly through other unknown mechanisms. Investigations are currently in progress to identify 4-HNE-modifed proteins involved in the NF-B signaling pathway as well as other possible mechanisms of 4-HNE-mediated suppression of NF-B activation.