Manganese superoxide dismutase: a hepatic acute phase protein regulated by interleukin-6 and glucocorticoids.

0013-7227/91/1295-2376$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 5 Printed in U.S.A.

Manganese Superoxide Dismutase: A Hepatic Acute Phase Protein Regulated by Interleukin-6 and Glucocorticoids* WILLIAM C. DOUGALLf AND HARRY S. NICK Department of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, University of Florida College of Medicine, Gainesville, Florida 32610

ABSTRACT. The superoxide dismutases (SODs) are important metallo-enzymes which scavenge and dismutate the superoxide free radical. They are thought to be the main enzymes in the antioxidant defense system. Identification of stimuli that control transcription of the SOD genes is essential for understanding SOD gene regulation. In this study we show that manganese SOD (MnSOD) mRNA levels are elevated by lipopolysaccharide, a bacterial endotoxin, in rat liver. However, neither lipopolysaccharide nor tumor necrosis factor-a had an effect on MnSOD mRNA expression in cultured primary hepatocytes. On the other hand, the inflammatory cytokines, interleukin-1 (IL-1) and IL-6 did increase MnSOD mRNA levels,

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NFLAMMATION causes an increase in many hepatic proteins termed acute phase reactants (APRs) (1-3). The majority of these hepatic proteins are targeted to the serum, where they act collectively to prevent cellular and tissue damage resulting from chronic inflammation. The production of hepatic APRs is elicited by inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-a (TNFa), and IL-6 (2, 4, 5). Together, during inflammation, these cytokines also induce fever, stimulate immunoglobulin production, activate T-cells, and cause tumor necrosis (1). We and others have shown that three inflammatory mediators, IL-1, TNFa, and lipopolysaccharide (LPS), also increase mRNA and protein levels of manganese superoxide dismutase (MnSOD) in pulmonary epithelial and endothelial cells (6-9). These results suggest that MnSOD may be an APR. This makes sense, because inflammation is known to cause oxidative damage to cells (2, 10). SODs, on the other hand, catalyze the dismutation of superoxide free radicals to hydrogen peroxide and water, and are considered to Received March 29,1991. Address all correspondence and requests for reprints to: Dr. Harry S. Nick, Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box J-245, Gainesville, Florida 32610. * This work was supported by NIH Grant RO1-KL39593 (to H.S.N.). t Current address: Division of Immunology, Department of Pathology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.

either 2- or 15-fold, respectively, over a 20-h period in hepatocytes. The IL-6-induced increase in MnSOD mRNA levels was attenuated by dexamethasone, a glucocorticoid, in hepatocytes cultured for less than 16 h. In contrast, in hepatocytes originally cultured for more than 16 h, IL-6 and dexamethasone produced a synergistic increase in MnSOD mRNA levels. The induction of MnSOD expression by IL-6, which is a known inflammatory cytokine, suggests that MnSOD may play a role in the inflammation process. Since inflammation is known to result in oxidative damage to cells, the role of MnSOD may be to protect cells from inflammation-mediated oxidative damage. (Endocrinology 129: 2376-2384, 1991)

be the primary enzymes of the antioxidant defense system. The liver plays a pivotal role in the acute phase response (1-3, 5). In this study we first investigated the effect of LPS on the expression of MnSOD mRNA in liver. We then evaluated the effects of LPS and other inflammatory cytokines on MnSOD mRNA expression in primary cultures of hepatocytes. Our results show that whereas LPS increases MnSOD mRNA levels in the liver, it has no effect on MnSOD mRNA levels in hepatocytes. Moreover, neither IL-1 nor TNFa increases MnSOD mRNA levels in hepatocytes. However, Northern analysis indicated that IL-6 specifically induced MnSOD mRNA expression in these cells. This IL-6dependent increase in MnSOD mRNA was attenuated by dexamethasone (DEX), which is known to reduce inflammation. However, attenuation of the IL-6 induction by DEX occurs only in hepatocytes cultured for less than 16 h. In hepatocytes cultured for longer times, DEX and IL-6 act synergistically to increase MnSOD mRNA expression. This result probably reflects intrinsic metabolic changes (11,12) in long term cultured hepatocytes, as also evidenced by changes in metallothionein mRNA expression.

Materials and Methods Male Sprague-Dawley rats (150-200 g) were injected ip with 750 Mg/kg BW S. thyphimurium LPS diluted into PBS, and RNA was isolated from liver tissue 24 h after injection.

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IL-6 INDUCTION OF MnSOD Hepatocyte isolation Rat hepatocytes were prepared by the collagenase perfusion technique originally described by Berry and Friend (13), as modified by Kilberg (14). Male Sprague-Dawley rats (150-200 g) were used as the source of hepatocytes. The viability of the hepatocytes was greater than 90%, as determined by trypan blue exclusion. Cells suspensions were washed in cold Dulbecco's Modified Eagle's Medium (DMEM) and plated on collagencoated plates at a density of 1 X 106 cells/ml in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were maintained at 37 C in 5% CO2 for 3 h. After this incubation, the medium was changed, and the experiment was initiated, as described in Results. RNA isolation and Northern analysis

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to LPS. Male Sprague-Dawley rats were injected with Salmonella typhimurium LPS, and total RNA was isolated from liver tissue 24 h later. Figure 1A shows the result of Northern analysis of this RNA. MnSOD mRNA was elevated in the liver tissue of two separate rats (lanes 2 and 3) relative to the control level (lane 1). Cu/ZnSOD and /?-actin mRNA served as internal standards to control for any loading errors from lane to lane (8). A quantitative summary of the induction of MnSOD mRNA standardized relative to Cu/ZnSOD mRNA levels A

Liver vf

Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method (15), as described previously (8). After Northern blotting, the membranes were hybridized for 12-18 h at 60 C with a 32P-labeled rat MnSOD, rat Cu/ZnSOD, j8-actin, human histone H4, or metallothioneinI and -II (MT-I and MT-II) probes. The MnSOD cDNA probe used for Northern hybridization was a 1000-basepair (bp) EcoRl/Pstl fragment derived from MnSOD cDNA containing the entire protein-coding region and 360 bp of the 3'-nontranslated portion, (Dougall, W. C, unpublished data). The rat Cu/ ZnSOD probe was a full-length cDNA isolated by this laboratory (Hsu, J. L., unpublished data) and identifies a 700-bp Cu/ ZnSOD mRNA on Northern analysis. The human /3-actin probe was obtained from Dr. Larry Kedes (Stanford University), and the human histone H4 probe was a kind gift of Drs. Janet and Gary Stein (University of Massachusetts). Oligonucleotide probes specific to the MT-I and -II genes were kindly provided by Dr. Robert Cousins (University of Florida) and prepared as previously described (16). The 32P-labeled cDNAs were either products of random primer extension (17) or were produced as single strand probes from M13 phage vector extension, as previously described (18). The membranes were washed in 0.04 M sodium phosphate and 0.1% sodium dodecyl sulfate at 65 C and then subjected to autoradiography using an intensifying screen at -85 C.

MnSOD

Cu/ZnSOD

Actin

Densitometry Cu/ZnSOD, MnSOD, and /3-actin mRNA levels were quantitated using the Bio Image Visage 60 video densitometry system (Millipore/Bio Image, Ann Arbor, MI), using whole band analysis software. All densitometry was presented as the sum of the intensity of all of the rat MnSOD bands. Data from individual bands were also analyzed, but are not presented, and were found to be similar to those for the sum of all bands. For all experiments, quantitative data were presented only on signals in the linear range of the film and the densitometer.

Results Endotoxin (LPS) elevates MnSOD mRNA levels in the livers of treated rats This experiment was designed to establish whether rats respond at the mRNA level to a systemic exposure

Control

LPS

FIG. 1. Northern analysis of RNA from rat livers treated with LPS. A, Male Sprague-Dawley rats (150-200 g) were injected ip with 750 fig/ kg BW S. tyuhimurium LPS, and RNA was isolated from liver tissue after 24 h. Lane 1 corresponds to RNA isolated from control (salinetreated) rat liver. Lanes 2 and 3 correspond to RNA isolated from liver tissue from two independent LPS-treated rats. Total RNA was isolated and analyzed by Northern hybridization using a 32P-labeled MnSOD, Cu/ZnSOD, or j8-actin cDNA probes. B, Quantitative image-processing data from MnSOD levels illustrated in A. Cu/ZnSOD, /3-actin, and MnSOD signals were quantitated by using the Bio Image Visage 60 video densitometry system (Millipore/Bio Image). Any variations in RNA loading were internally controlled by assuming that Cu/ZnSOD mRNA levels were unchanged with respect to experimental conditions. Individual lanes were then compared with respect to the intensity of the Cu/ZnSOD signal, i.e. the data are expressed as a ratio of MnSOD to Cu/ZnSOD. Relative induction is expressed as the mean of the two experiments shown in A relative to the control value. The induction is also representative oi" data from six other animals.


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IL-6 INDUCTION OF MnSOD

is shown in Fig. IB. There was about a 6-fold increase over the control value. Actin mRNA levels were high in LPS-treated liver. Nevertheless, quantitative densitometric analysis of the MnSOD based on the actin data gave a 2.5-fold relative increase in the MnSOD (data not shown).

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Endotoxin (LPS) has no effect on hepatocyte MnSOD mRNA levels

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Primary cultures of hepatocytes are well characterized with respect to normal metabolic processes (12, 13, 19), including responsiveness to IL-1, TNFa, and IL-6 (20), and have been extensively used in the study of the acute inflammatory response. For the experiments described below, isolated hepatocytes were incubated for 3 h to allow attachment to the collagen matrix. In this study we define short term cultured (STC) hepatocytes as hepatocytes treated with the various regimens immediately after the initial 3-h plating. Long term cultured (LTC) hepatocytes are plated for 3 h, as were the STC hepatocytes, and then cultured without treatment for 16 h, after which time the experimental treatments were initiated. This distinction between short term and long term cultured hepatocytes will be relevant in the discussion of IL-6 and the effects of steroids on MnSOD expression. The effect of LPS on MnSOD gene expression was measured in primary cultures of STC rat hepatocytes to determine whether the in vivo LPS effects (Fig. 1) could be mimicked in vitro. Surprisingly, Northern analysis of RNA isolated from STC hepatocytes showed no increase in MnSOD mRNA abundance after LPS treatment, as shown in Fig. 2A. This is in contrast to pulmonary epithelial cells (L2 cells) treated with LPS (8). Under similar conditions these cells underwent a 15- to 20-fold increase in MnSOD mRNA even at concentrations of LPS that were 100 times smaller than the highest level used on hepatocytes. A quantitative summary of the induction of MnSOD mRNA standardized relative to Cu/ZnSOD mRNA levels is shown in Fig. 2B. The difference in basal level expression in the liver (Fig. 1, lane 1) vs. that in the hepatocytes is addressed in Fig. 3. Basal and cytokine-mediated hepatocyte MnSOD expression We examined steady state MnSOD mRNA by Northern analysis to establish basal MnSOD expression in STC hepatocytes as a function of culture time (Fig. 3). Five MnSOD mRNAs were detected by hybridization of total RNA from hepatocytes with a rat MnSOD cDNA probe. These were transcribed from a single MnSOD gene and most likely generated via differential polyadenylation (Dougall, W. C, unpublished data). The Northern analysis illustrated in Fig. 3 shows that basal MnSOD

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Endo•1991 Vol 129 • No 5

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Endotoxin L2 HEPATOCYTES FIG. 2. Northern analysis of RNA isolated from pulmonary epithelial cells and hepatocytes exposed to LPS. A, The pulmonary epithelial cells (L2) were grown to 90% confluency then exposed to 0.5 ng/ral S. typhimurium LPS for 12 h. Primary cultures of rat hepatocytes were prepared as described and subsequently exposed to S. typhimurium LPS. Total RNA was isolated and analyzed by Northern hybridization using 32P-labeled Mn- and Cu/ZnSOD cDNAs. Lane 1 corresponds to control L2 cells, whereas lane 2 represents RNA isolated from L2 cells exposed to LPS. Lane 3 corresponds to RNA isolated from control hepatocytes. Lanes 4,5, and 6 represent RNA isolated from hepatocytes exposed for 12 h to 1, 10, and 100 ng/ral S. typhimurium LPS, respectively. These autoradiograms illustrate representative data for at least five experiments for each cell type. B, Quantitative image processing of data for MnSOD levels illustrated in A These data were normalized and calculated as described in Fig. IB.

mRNA levels increased with the incubation time of the hepatocytes, apparently achieving maximum levels after 12 h of incubation. However, actinomycin-D inhibited this increase, such that MnSOD mRNA concentrations remained at the same basal level. The same membrane hybridized with a radiolabeled Cu/ZnSOD cDNA fragment confirmed that uniform amounts of RNA were present in each lane. Inflammatory cytokine IL-6 elevates MnSOD mRNA levels in hepatocytes Since LPS did not elevate MnSOD mRNA in hepatocytes, it is possible that in liver the effects of LPS may not be direct, but, rather, reflect a response to other LPS-induced inflammatory mediators secreted by other


IL-6 INDUCTION OF MnSOD Time (hr)

0

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Cu/ZnSOD 1 2 3 4 5 6 7 8 910 11 12 13 FIG. 3. Effect of plating time on basal MnSOD mRNA expression. STC hepatocytes were incubated for up to 24 h with or without 4 jtM actinomycin-D. Total RNA was isolated at the time points indicated and analyzed by Northern analysis using 32P-labeled MnSOD and Cu/ ZnSOD cDNA fragments, as described in Materials and Methods. The Northern analysis illustrated is representative of three independent experiments.

IL-1o TNF-a IL-6 FIG. 4. Northern analysis of hepatocytes exposed to IL-la, TNFa, and IL-6. Primary cultures of rat hepatocytes were exposed to 2 ng/ml ILla, 10 ng/ml TNFa, or 100 HSF units/ml IL-6 for 12 h. Total RNA was then isolated and evaluated by Northern hybridization to a 32Plabeled MnSOD or Cu/ZnSOD cDNA. Quantitative densitometric data of MnSOD mRNA levels shown appear as bar graphs. Any variations in RNA loading were internally controlled by assuming that Cu/ZnSOD mRNA levels were unchanged with respect to experimental conditions. The fold induction is calculated relative to control (CT) MnSOD mRNA levels at 12 h. The relative induction for MnSOD after IL-la and IL-6 treatment is 2.1- and 5.2-fold, respectively. This Northern analysis is representative of three independent experiments.

resident liver cells (1). To test this possibility, hepatocytes were exposed to IL-la, TNFa, and IL-6 for 12 h. Total RNA was then isolated and analyzed by Northern hybridization, and the densitometric summary of these data is shown in Fig. 4. The results indicate that TNFa had no effect, IL-la caused a 2-fold induction, and IL-6 elevated significantly the level of MnSOD mRNA in hepatocytes. We have, therefore, chosen to focus our efforts on the regulation of MnSOD by IL-6. The increase in MnSOD mRNA is a function of IL-6 concentration, as shown in Fig. 5. The maximal increase of 7-fold in MnSOD mRNA occurred with a concentration of 100 hepatocyte-stimulating factor (HSF) units/ ml IL-6. Figure 6 shows the kinetics of IL-6-mediated increases in MnSOD mRNA. After 2 h of incubation,

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0 .001 .01 .1 1 10 100 1000 IL-6 (units/ml) FIG. 5. IL-6 dose-response curve. Hepatocytes were exposed to increasing concentrations of human recombinant IL-6 for 12 h and evaluated by Northern analysis using a 32P-labeled MnSOD and Cu/ZnSOD cDNAs. The graph illustrates the level of MnSOD expression in hepatocytes after treatment with 0, 103, 10"2, 1 0 \ 1.0, 10, 100, and 1000 HSF units/ml recombinant human IL-6. The graph illustrates the percent maximal induction for the IL-6 concentrations described in Fig. 3 based on quantitative densitometric data. The 100 U/ml value was determined to be the 100% maximal induction of MnSOD mRNA levels, and the effects of other concentrations were plotted relative to this value. The densitometric values were determined for two Northern blots for all IL-6 concentrations, except 100 U/ml, for which n = 7, and are plotted as the mean ± SD.

MnSOD mRNA increased to 40% of its maximal level and approached its maximal amount by 24 h. There was no change in Cu/ZnSOD mRNA levels in response to IL-6 over 36 h, again showing that Cu/ZnSOD mRNA is a good internal control. Glucocorticoids enhance the stimulatory effect of IL6 on the synthesis of many, but not all, acute phase proteins (21-24). Moreover, the presence of glucocorticoids is stringently required for the IL-6 regulation of other acute phase proteins (25, 26). Thus, we examined the effect of DEX, a synthetic glucocorticoid, on basal and IL-6-induced MnSOD expression. STC rat hepatocytes were incubated with DEX, IL-6, or DEX plus IL6. At 2, 6, 12, and 24 h, culture samples were removed and analyzed by Northern hybridization. Treatment with IL-6 resulted in the usual increase in MnSOD expression (Figs. 7). Treatment with DEX alone caused a slight inhibition of MnSOD expression. However, treatment with DEX and IL-6 produced unusual results. This combination of compounds markedly attenuated MnSOD expression in cells incubated up to 12 h. In contrast, in cells incubated for 24 h, IL-6 plus DEX acted synergistically to increase MnSOD expression. This treatment resulted in a 20-fold induction over that in control cells or cells treated with DEX alone. We also determined MT mRNA levels as a positive internal control for the actions of DEX and IL-6, since MT production is known to be stimulated by these agents (16, 27). As shown in Fig. 7A, DEX treatment elevated levels of MT mRNA at 6, 12, and 24 h (lanes 8, 12, and 16). IL-6 treatment alone caused an increase in MT


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TIME (hr) FIG. 6. Time course of induction of MnSOD mRNA by IL-6. A, Hepatocyte primary cultures were incubated for up to 36 h with DMEM10% FBS or DMEM-10% FBS with 100 U/ml IL-6. At the time points indicated, UNA was isolated and analyzed by Northern analysis using radiolabeled MnSOD and Cu/ZnSOD cDNAs. The Cu/ZnSOD control indicated that an equivalent amount of RNA had been loaded in each lane. B, The Cu/ZnSOD and MnSOD mRNA signals were quanitated by densitometry, and the percent maximal induction of the MnSOD from IL-6-treated cells is plotted relative to the control value at each time point. The densitometric values were determined for two separate Northern blots for the graph illustrated here and are plotted as the mean ± SD. The Northern analysis shown is representative of seven individual experiments.

mRNA levels at 2, 6, and 12 h (lanes 3, 7, and 11), but not at 24 h (lane 14). Treatment with both IL-6 and DEX had an additive effect on MT mRNA levels at all times. We then investigated why DEX and IL-6 did not synergistically increase MnSOD mRNA levels at the earlier times, i.e. up to 12 h. It is possible that some component in the serum could be involved in preventing the action of DEX at the early times, as shown in Fig. 7A (lanes 5, 9, and 13). To test this, a modification of the experiment above was repeated in which FBS was present or absent during incubation of STC or LTC hepatocytes. Figure 8 shows that FBS had no effect on either STC or LTC hepatocytes and confirms the synergistic effect of IL-6 and DEX in LTC cells. As a positive internal control we also measured MT mRNA levels. As seen in Fig. 8, A and B, MT expression was independently enhanced in STC hepatocytes by IL-6 and DEX. In combination, IL-6 and DEX additively increased MT

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(hours) FIG. 7. Effects of glucocorticoids on IL-6-induced MnSOD expression. A, Primary cultures of rat hepatocytes were incubated with DEX (1 MM), IL-6 (100 U/ml), or DEX plus IL-6 for 0-24 h. The RNA was isolated at the time points indicated and analyzed by Northern analysis using radiolabeled MnSOD, Cu/ZnSOD, and MT probes. The internal control indicated that an equivalent amount of total RNA was present in each lane. B, Quanitative densitometric data of the MnSOD mRNA levels from A. The Northern analysis shown is representative of six independent experiments.

mRNA in STC cells (Fig. 8, A and B). In contrast, in LTC hepatocytes (Fig. 8, C and D) DEX caused a small increase in MT mRNA, while IL-6 had no effect, but together they act synergistically to markedly increase MT mRNA (16). Effect of actinomycin-D and cycbheximide on IL-6induced MnSOD expression The increased levels of MnSOD steady state mRNA after IL-6 treatment could be a reflection of increased transcription of the mRNA, increased stability of the message, or a combination of both. To address the potential transcriptional mechanism for this increase in steady state levels of mRNA, STC hepatocyte cultures were incubated for 16 h with IL-6, 4 /iM actinomycin-D (a potent RNA synthesis inhibitor), or both IL-6 and actinomycin-D. Figure 9 illustrates that incubation with actinomycin-D alone reduced the basal levels of MnSOD (lane 3), as was shown at similar time points in Fig. 3. However, cotreatment of hepatocytes with IL-6 and actinomycin-D completely abolished the IL-6-dependent



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FIG. 8. Effects of serum and glucocorticoids on IL-6-induced MnSOD expression in STC and LTC hepatocytes. A, Primary (STC) cultures of rat hepatocytes were cultured in either DMEM lacking 10% FBS (lanes 1, 2, 3, and 4) or DMEM supplemented with 10% FBS (lanes 5, 6, 7, and 8). In addition, the cells were simultaneously cultured under the following conditions: control (lanes 1 and 5), 100 U/ml IL-6 (lanes 2 and 6), 1 nM DEX (lanes 3 and 7), or 100 U/ml IL-6 and 1 juM DEX (lanes 4 and 8). Total cellular RNA was isolated after 16 h of these treatments and analyzed by Northern analysis using radiolabeled MnSOD, Cu/ZnSOD, /9-actin, and MT probes. B, Quanitative densitometric analysis of the MnSOD mRNA levels from A. The Northern analysis shown is representative of three independent experiments. C, Primary (LTC) cultures of rat hepatocytes were isolated as described in Materials and Methods. Cells were plated in DMEM or DMEM supplemented with 10% FBS and cultured for 16 h without experimental treatment. The medium was decanted and replaced with fresh DMEM containing 10% FBS when indicated, and treated with 100 HSF U/ml IL-6,1 MM DEX, or IL-6 plus DEX. Total cellular RNA was isolated 16 h later and analyzed by Northern analysis with radiolabeled MnSOD, Cu/ZnSOD, /3-actin, and MT probes. D, Quanitative densitometric analysis of the MnSOD mRNA levels from C. The Northern analysis shown is representative of three independent experiments.

increase in MnSOD mRNA (lane 4). The same membrane hybridized with a radiolabeled Cu/ZnSOD cDNA indicated that each lane contained uniform levels of total RNA. Histone H4 mRNA levels were also evaluated in this experiment to determine the efficacy of actinomycinD (Fig. 9, lanes 3 and 4), since histone transcription is inhibited by actinomycin-D (8). To determine whether de novo protein synthesis is required for the induction of MnSOD mRNA by IL-6, STC hepatocytes were cotreated with IL-6 and cycloheximide (a protein synthesis inhibitor), and RNA was isolated after 16 h of treatment. Incubation of cells with 50 /iM cycloheximide alone marginally reduced the basal

expression of MnSOD mRNA (Fig. 9, lane 5). When cells were treated with both IL-6 and cycloheximide, steady state levels of MnSOD were elevated relative to those in cells treated only with cycloheximide (Fig. 9, lanes 5 and 6). Rehybridization of this membrane with a Cu/ZnSOD cDNA probe again indicated that equivalent amounts of RNA were loaded in each lane.

Discussion Mononuclear phagocytes (and other cells) synthesize and release the cytokines IL-1, TNF, and IL-6, which initiate the acute phase inflammatory response (28-30).



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Interleukin-6 —

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Actinomycin D — — Cycloheximide — —

MnSOD

Cu/ZnSOD

Histone

1 2

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FIG. 9. Effects of actinomycin-D and cycloheximide on IL-6 induction of MnSOD mRNA. Total RNA was isolated from hepatocytes under control conditions (lane 1) or after exposure to 100 U/ml IL-6 (lane 2), 4 nM actinomycin-D (lane 3), both IL-6 and actinomycin-D (lane 4), 50 nM cycloheximide (lane 5), or both IL-6 and cycloheximide (lane 6) for 16 h. The RNA was evaluated by Northern analysis using 32Plabeled MnSOD, Cu/ZnSOD, and histone H4 cDNAs. The left panel illustrates the actinomycin experiment, and the right panel illustrates the cycloheximide experiment. These data are representative of three independent experiments.

Administration of LPS, IL-1, or TNF to pulmonary epithelial cells also mediates an increase in MnSOD mRNA at the transcriptional level (8), analogous to other acute phase proteins. In the present study we have extended these studies to examine the effects of inflammatory cytokines on hepatic MnSOD expression in both liver and hepatocytes, given the importance of liver in the inflammatory process. We first showed that LPS injected into rats caused an increase in liver MnSOD mRNA. Hepatocytes in primary culture do not respond similarly. This suggests that the LPS response in liver may not be direct, but, rather, reflects a secondary response to some other LPS-induced inflammatory mediator. To test this hypothesis, primary cultures of hepatocytes were treated in vitro with various inflammatory cytokines, and MnSOD mRNA was measured by Northern analysis. First, it was necessary to determine basal levels of MnSOD mRNA in cultured hepatocytes. Figure 3 shows that MnSOD mRNA levels increase with increasing periods of culture. This increase is inhibited by actinomycin and probably reflects a transcriptional induction of the gene associated with culturedependent hepatocyte changes. Having established that hepatocytes contain significant concentrations of MnSOD mRNA, we next treated them with three inflammatory cytokines: IL-1, TNFa,

Endo• 1991 Voll29«No5

and IL-6. As shown in Fig. 4, only IL-6 and IL-1 caused an increase in MnSOD mRNA; LPS and TNFa were without effect. These observations support the idea that liver may respond to systemic LPS via LPS stimulation of IL-6 and/or IL-1 secretion or perhaps by increasing the number of their respective receptors. They also establish the utility of primary cultures of rat hepatocytes as an in vitro model system to study the regulation of MnSOD by IL-6. This is the central topic of this paper. We next showed that the increase in MnSOD mRNA is a function of IL-6 concentration, and we found 100 U/ ml to be optimal. The kinetics of MnSOD RNA production indicate that about 6 h of exposure to IL-6 are required for half-maximal induction. Glucocorticoids and IL-6 synergistically increase the synthesis of most, but not all, APRs (16, 21, 22, 24, 26). DEX and IL-6 also synergistically increase MnSOD mRNA, but only after hepatocytes (LTC hepatocytes) have been cultured longer than 16 h. Hepatocytes (STC hepatocytes) cultured for less than 16 h do not respond to this synergistic effect of DEX and IL-6. We note that IL-6 treatment of STC hepatocytes resulted in about a 4-fold increase in MnSOD mRNA levels compared to about a 2-fold increase in LTC hepatocytes. This points further to the difference between LTC and STC hepatocytes. The results in Fig. 8 also show that whatever this difference may be, it is not due to some component in the FBS present in the culture medium. In these experiments we also probed for MT mRNA as a positive control to verify the effectiveness of DEX in this system. This is because MT expression is known to be stimulated by DEX (16, 27). We, likewise, observed this stimulation, but it was greatly reduced in LTC hepatocytes. Moreover, IL-6 and DEX act synergistically to increase MT mRNA (16) in LTC hepatocytes, analogous to the way they increased MnSOD mRNA. Thus, the difference between STC and LTC hepatocytes is not specific to MnSOD message, but, rather, reflects some intrinsic factor that changes as the cells are cultured for long periods of time (14). These results indicate that culture conditions may be important in these types of experiments. Glucocorticoid IL-6 synergy documented for other APRs (16, 21, 22, 24) is usually observed under culture conditions that would qualify the cells as LTC hepatocytes. The question arises of whether the synergy seen in these experiments also is due to the culture conditions, and if so, why? One hypothesis that could explain the data is that a down-regulation in the number or affinity of IL-6 receptors occurs as the hepatocytes begin to lose properties characteristic of the liver in vivo (31). This hypothesis is also consistent with recent studies which indicate that the synergistic effect of IL-6 and DEX on acute phase gene transcription is mediated by a glucocorticoid-dependent stimulation of IL-6 receptor



synthesis (32). The researchers in that study have shown that induction of IL-6 receptor expression requires 14 h of DEX treatment, which could explain the potentiation of IL-6-induced MnSOD mRNA by DEX after 16 h. Likewise, the requirement for DEX in the induction of MT by IL-6 in LTC hepatocytes is consistent with the above hypothesis (16). We used inhibitors of RNA and protein synthesis to characterize further the mechanism by which IL-6 itself induces MnSOD mRNA levels. The IL-6-mediated increase was abolished by actinomycin-D, an RNA synthesis inhibitor, indicating that this increase in MnSOD mRNA is dependent upon transcription. Cycloheximide, a protein synthesis inhibitor, did not abolish the IL-6mediated increase in MnSOD, suggesting that this induction does not rely on de novo protein synthesis. These data suggest a model for the IL-6 induction of MnSOD steady state mRNA levels that involves a direct transcriptional activation of the MnSOD gene. Furthermore, because de novo protein synthesis is not required, the increased rate of transcription could be mediated through an existing transcription factor, which is activated by IL-6 through a posttranslational modification, thereby affecting the MnSOD promoter. IL-6 mediates the transcriptional activation of a2macroglobulin (33) and other acute phase proteins (20, 24, 34-36). Transfection experiments with promoter mutants have identified cis-acting response elements (IL6REs) in the promoter of the a2-macroglobulin gene and in the promoters of other acute phase genes (26, 32, 3638). We have, likewise, identified two potential IL-6REs in the rat MnSOD gene promoter (Dougall, W. C, unpublished data). There is also mounting evidence for nuclear factors that interact with IL-6REs (33, 37, 39). It is possible that transcription of the MnSOD gene is also regulated by IL-6 via IL-6REs and associated nuclear factors. Exposure of animals to LPS results in the release of O2~ from phagocytes (10). This effect, mediated by complement activation, may damage mitochondria directly during inflammation. Several observations support this hypothesis. Oxidative metabolism is defective in animals suffering from endotoxic shock. LPS destroys the mitochondrial proton gradient in cells and leads to the accumulation of superoxide and peroxides (40). LPS induces hemorrhagic necrosis of sensitive tumors and uncouples oxidative phosphorylation (41, 42). LPS-activated cytotoxic macrophages inhibit mitochondrial electron transport in lymphoblastic leukemia cells (43). Because of the considerable oxidative stress to the mitochondria during inflammation, the physiological role of MnSOD may be to protect mitochondria against free radicals generated from inflammatory processes. Our data showing that MnSOD is induced by inflammatory mediators targeted

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to specific tissues support this hypothesis. Furthermore, the induction of MnSOD by all of the acute phase cytokines demonstrates that this mitochondrial-localized protein is a vital member of the acute phase reactants.

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