50% deionized formamide/0.25 M sodium phosphate, pH. 7.2/0.25 M NaCl/1 mM ..... Sidney M. Morris, Ph.D., and David J. Tweardy, M.D., of the. University of .... Sheehan, K. C. F. & Schreiber, R. D. (1992) in Tumor Necro- sis Factors, The ...
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 522-526, January 1993
Cell Biology
Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes DAVID A. GELLER*t, ANDREAS K. NUSSLER*, MAURICIo Di SILVIO*, CHARLES J. LOWENSTEINt, RICHARD A. SHAPIRO*, STEWART C. WANG*, RICHARD L. SIMMONS*, AND TIMOTHY R. BILLIAR* *Department of Surgery, 497 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261; and tDepartment of Neuroscience, Johns Hopkins Medical Institutions, Baltimore, MD 21205
Communicated by Klaus Hofmann, September 18, 1992
rophage NOS has been cloned from RAW264.7 cells by three groups and identifies an -4.4-kb mRNA (12-14). The physiologic importance of NON as a vasodilator, neurotransmitter, and antimicrobial/antitumor agent is rapidly becoming apparent. Previous work showed that rat hepatocyte/Kupffer cell cocultures stimulated with lipopolysaccharide (LPS) produce large amounts of nitrite (NO-) and nitrate (NO-), the stable end products of the NO- pathway (15). Further, it was demonstrated that hepatocytes also produce NO- in vivo during chronic hepatic inflammation (16, 17) and in vitro in response to conditioned Kupffer cell supernatant (18) or to a mixture of LPS and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (19). Human hepatocytes were also stimulated to produce NO' by the same combination of endotoxin and cytokines as rat hepatocytes (20). However, essentially nothing has been
ABSTRACT Nitric oxide (NO-) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO' synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon v (IFN-y). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-y. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at --4.5 kilobases which was maximally up-regulated ('70-fold) by the combination of TNF, IL-1, IFN-y, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-y. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginlne had no effect on mRNA levels but effectively blocked NO- formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytpkines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.
reported about the direct signals required for inducible NOS gene activation. Therefore, the present study was undertaken to characterize the molecular regulation of the inducible rat hepatocyte NOS by endotoxin and cytokines known to
up-regulate hepatocyte NO' synthesis. MATERIALS AND METHODS Isolation of Hepatocytes. Hepatocytes were isolated from male rats (200-250 g, Harlan-Sprague-Dawley) by a modification of the in situ collagenase (Sigma) perfusion technique of Seglen (21). Hepatocytes were separated from nonparenchymal cells by differential centrifugation at 50 x g and then passed over a 30%o Percoll gradient to obtain a highly purified cell population. Hepatocyte purity assessed by microscopy was >98% and viability consistently exceeded 95% by trypan blue exclusion. For the in vivo studies, hepatocytes were harvested from rats 3 days after injection of Cornyebacterium parvum (Wellcome Biotechnology; 28 mg/kg, i.v.) and from normal rats as control. Cell Culture. Hepatocytes (5 x 106 in 6 ml of medium) were plated onto 100-mm gelatin-coated Petri dishes (Coming). Medium consisted of Williams' medium E (GIBCO) with L-arginine (0.50 mM), insulin (1 1LM), Hepes (15 mM), L-glutamine, penicillin, streptomycin, and 10% lowendotoxin calf serum (HyClone). After a 24-hr incubation, the medium was changed to include a cytokine mixture (CM) containing LPS (Escherichia coli 0111:B4, Sigma; 10 ,ug/ml), human recombinant IL-1p8 (Cistron, Pine Brook, NJ; 5 unit/ ml), TNF (Genzyme; 500 units/ml), and IFN-y (Amgen; 100 units/ml) or various combinations of LPS and cytokines. Other experimental conditions included addition of cytokines with dexamethasone, actinomycin D, or cycloheximide (all
Following the discovery of the nitric oxide (NO-) pathway and its identification as endothelium-derived relaxing factor, a variety of cell types such as macrophages (1, 2), endothelial cells (3, 4), smooth muscle cells (5), and neurons (6, 7) have been shown to produce NO' from L-arginine. Constitutive and inducible isoforms of the NO' synthase (NOS) enzyme exist, and they differ in structure and regulation (8). The neuronal constitutive NOS is a 150-kDa protein whose activity is dependent upon calcium and calmodulin (7); the inducible macrophage NOS is a 130-kDa protein which is thought to function independently of calcium/calmodulin (9, 10). The constitutive NOS cDNA has been cloned from rat cerebellum and identifies an ==10-kilobase (kb) mRNA on Northern blot analysis (11), while the inducible murine mac-
Abbreviations: NO-, nitric oxide; NOS, NO' synthase; NMA, NGmonomethyl-L-arginine; CM, cytokine mixture; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL-1, interleukin 1; IFN-y, interferon fy. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 522
Cell Biology: Geller et al. Sigma) or NG-monomethyl-L-arginine [NMA, prepared as described (17)]. After cultures were incubated at 370C in 95% air/5% CO2 for 2-24 hr, supernatants were collected for NO-2 + NO3- and cGMP assays, and total RNA was extracted for Northern blot analysis. After collection, supernatant samples for cGMP assay had the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) added and all samples were stored at -700C until used. NOj + NO- and cGMP Measurement. To determine the amount of NO- produced by hepatocytes, the culture supernatants were assayed for the stable end products of NOoxidation, NO- and NO3-, by an automated procedure based on the Griess reaction (15). cGMP levels were measured with a commercially available radioimmunoassay according to the manufacturer's instructions (NEN). Molecular Probes. The inducible-NOS probe used was a mouse macrophage cDNA clone (14). Not I digestion of pBluescript plasmid containing a fragment of the cloned mouse NOS cDNA yielded a 2.7-kb cDNA insert which was found to effectively identify the rat hepatocyte NOS mRNA on cross-species hybridization (see Results). After hybridization with the NOS probe, Northern blot membranes were stripped with boiling 5 mM EDTA/0.1% SDS and rehybridized with a probe specific for 18S rRNA to control for variations in amount of RNA per lane. RNA Isolation and Northern Blot Analysis. Total RNA was extracted from freshly isolated or cultured hepatocytes using the RNAzol B (Biotecx Laboratories, Houston) modified method of Chomczynski and Sacchi (22). All RNA samples had an A260/A280 ratio > 1.50. Aliquots containing 20 ug of total RNA were electrophoresed in 1% agarose gel containing 3% formaldehyde. RNAs were blot-transferred to GeneScreen membrane (NEN) and UV autocrosslinked. Membranes were hybridized with the probe overnight at 43°C in 50% deionized formamide/0.25 M sodium phosphate, pH 7.2/0.25 M NaCl/1 mM EDTA/7% SDS containing denatured salmon sperm DNA (100 ,ug/ml). DNA probes (2-4 x 106 cpm/ml) were labeled with [a-32P]dCTP (specific activity, 3000 Ci/mmol; NEN; 1 Ci = 37 GBq) by random priming. The hybridized filters were washed at 53°C in 2x standard saline citrate (SSC)/0.1% SDS/25 mM sodium phosphate/i mM EDTA/0.1% SDS and finally in 25 mM sodium phosphate/1 mM EDTA/1% SDS. Autoradiography was performed by exposure (2 hr to 11 days) to Kodak X-Omat film at -70°C in the presence of intensifying screens. Relative mRNA levels were quantitated by scanning densitometry. Each Northern blot shown is representative of at least three separate experiments performed at different times, unless otherwise specified. Statistical Analysis. Values for NOj + NO3- and cGMP are means ± SEM. The significance of differences was determined with a two-tailed Student's t test. Statistical significance was established at a P value < 0.05.
RESULTS Fig. 1 shows the time course ofNOS induction in hepatocytes in vitro following exposure to LPS, TNF, IL-1, and IFN-,y, a combination referred to here as CM. Cultured rat hepatocytes were stimulated (S) with CM in vitro for 2-24 hr and were compared with unstimulated (U) hepatocytes. Northern blot analysis revealed up-regulation of hepatocyte NOS mRNA levels by CM stimulation (Fig. 1A). The mRNA was seen as a single band at -4.5 kb which was faintly present 2 hr after stimulation, became maximal at 8 hr. and then declined slightly by 24 hr (Fig. 1B). In the unstimulated hepatocytes, NOS mRNA was not detectable. To compare NOS mRNA levels with NOS activity, we determined hepatocyte NO- release by measuring culture supernatant NO- + NO3- levels (Fig. 1C). Increases in hepatocyte nitrogen oxide
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levels were first seen at 8 hr after CM stimulation, with significant further increases to 86.1 juM at 12 hr and to 284.6 ,uM at 24 hr. To determine whether the individual stimuli would induce NOS mRNA expression, and if so, whether they would act in a similar time-dependent manner, experiments were performed using the single agents at concentrations previously found to optimally induce hepatocyte NOS activity (19). The peak induction of NOS mRNA was seen at 4 hr after stimulation with TNF or IFN-y, whereas IL-1 induced a peak at 8 hr (Fig. 2). No mRNA was detected with LPS stimulation, nor in the unstimulated hepatocytes. Unlike CM stimulation, where mRNA levels remained 75% of maximal at 24 hr, with any single cytokine the NOS mRNA was decreased by 12 hr following stimulation and was nearly gone at 24 hr. This suggests that the cytokine effects are additive or synergize to produce a prolonged induction of NOS mRNA.
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Twenty-four hours after stimulation, only TNF (11.5 ,uM) and, to a lesser extent, IL-1 (7.6 ,uM) induced appreciable rises in NO- + NOj levels (Fig. 2). These levels are relatively low ( IL-1 > IFN-y > LPS. TNF was the most effective single cytokine, achieving 10%o of the maximal (CM-induced) hepatocyte NOS mRNA level. LPS alone was the weakest stimulator, with a trace mRNA signal after prolonged autoradiographic exposure ( IL-1 + IFN-y >> LPS + TNF > LPS + IL-1 > LPS + IFN-y. TNF + IFN-y or TNF + IL-1 was most effective, achieving 35-45% of maximal induction. Clearly, the least effective double combinations were LPS with any cytokine, producing only 7-15% of maximal levels. For triple cytokines, LPS + TNF + IFN-y - TNF + IL-1 + IFN-y > LPS + IL-1 + IFN-y > LPS + TNF + IL-1. The most effective triple cytokine combinations were LPS + TNF + IFN-y and TNF + IL-1 + IFN-y, achieving 65-75% of maximal induction. To correlate mRNA levels with NOS activity, NOj + NO3 levels were measured in the culture supernatants collected at
FIG. 3. Comparison of LPS and cytokine combinations in stimulating hepatocyte NOS mRNA expression. Cultured rat hepatocytes were stimulated for 6 hr with various combinations of LPS and cytokines. (A) Effect of single vs. double signal combinations in up-regulating hepatocyte NOS mRNA. (B) Effect of double vs. triple signal combinations in up-regulating hepatocyte NOS mRNA.
the time of RNA harvesting (Table 1). Culture supernatants from unstimulated (control) hepatocytes had a basal concentration of4.1 ,uM NO- + NO-. This rose to 28.9 uM 6 hr after CM addition and was reversed by the addition of NMA. In Table 1. Rat hepatocyte nitrogen oxide and cGMP production 6 hr after in vitro stimulation fmol per NO- + NOT, cGMP, Group ,uM 105 hepatocytes Control 4.1 ± 0.63 103.1 ± 8.2 CM 28.9 ± 5.40* 282.0 ± 37.6* CM + NMA 4.2 ± 0.92t 138.4 ± 34.8t LPS 2.5 ± 0.35 164.0 ± 52.5 TNF 8.9 ± 3.52* 262.0 ± 145.2 IL-1 4.5 ± 1.39 145.2 ± 36.7 4.5 ± 0.35 158.0 ± 41.2 IFN-y LPS + TNF 9.6 ± 2.30* 202.2 ± 66.2* LPS + IL-1 4.1 ± 0.70 125.4 ± 38.1 LPS + IFN-y 4.1 ± 0.50 119.2 ± 40.0 TNF + IL-1 14.6 ± 3.70* 317.8 ± 80.3* TNF + IFN-y 15.7 ± 5.30* 319.2 ± 133.7* IL-1 + IFN-'y 10.4 ± 3.15* 266.4 ± 106.1* LPS + TNF + IL-1 13.9 ± 2.60* 266.2 ± 86.1* LPS + TNF + IFN-y 19.4 ± 3.00* 302.8 ± 75.5* LPS + IL-1 + IFN-y 10.3 ± 2.35* 251.2 ± 82.8* TNF + IL-1 + IFN-y 22.4 ± 5.75* 280.8 ± 65.3* *P < 0.05 vs. control. t, P < 0.05 vs. CM.
Cell Biology: Geller et al.
Proc. Natl. Acad. Sci. USA 90 (1993)
agreement with the mRNA levels, TNF as a single agent most effectively induced NOS activity. From the double signal combinations, TNF with IFN-y or IL-1 stimulated the highest nitrogen oxide synthesis (NOr + NO3 concentrations of 15.7 and 14.6 1LM, respectively), again correlating very well with mRNA levels. A similar association between mRNA levels and NO- + NOj synthesis was seen with the triple cytokine combinations. Soluble guanylate cyclase activation is a sensitive indicator of NO synthesis, and much of the cGMP produced by cultured hepatocytes is released into the medium (23). Therefore, cGMP levels were used as an additional marker of increased NO formation. Increased extracellular cGMP coincided with increased NO- + NO- (Table 1). The addition of NMA reversed the rise in cGMP, and no combination of single, double, or triple cytokines and LPS increased cGMP without also increasing nitrogen oxide release. Taken together, the results are consistent with previous findings associating hepatocyte NO- with cGMP synthesis (23). Glucocorticoids decrease NO- synthesis in vitro in endothelial cells (24) and macrophages (25). Therefore, we investigated the effects of dexamethasone on CM-induced hepatocyte NOS mRNA levels (Fig. 4). Dexamethasone decreased NOS mRNA levels by 36% at 10-8 M, and by 63% at 10-6 M. The addition of the protein synthesis inhibitor cycloheximide also decreased NOS mRNA levels in a dosedependent manner, whereas the transcriptional inhibitor actinomycin D blocked NOS mRNA induction. These agents did not decrease hepatocyte viability determined by trypan blue exclusion (data not shown). The addition of NMA effectively inhibited NO- synthesis (Table 1) but had no effect on cytokine-induced mRNA levels for hepatocyte NOS. To detect basal levels of NOS mRNA in unstimulated hepatocytes, longer autoradiographic exposures of Northern blots were required (11 days). Fig. 5 shows very low basal levels of NOS mRNA in unstimulated hepatocytes and permits a measurement of fold induction in the CM-stimulated cells. Averaging the densitometry values of four different rat hepatocyte cultures indicated that CM stimulation produced =70-fold induction of NOS message. To compare the in vitro induction of hepatocyte NOS by CM with in vivo induction, rats were injected with heat-killed C. parvum, producing chronic hepatic inflammation and hepatocyte NO- biosynthesis (16). Fig. 6 shows both in vivo induction of hepatocyte NOS mRNA 3 days after C. parvum injection and in vitro induction by CM stimulation. After x
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DISCUSSION In this report, we describe the regulation of the inducible hepatocyte NOS by LPS, cytokines, and dexamethasone. Using a mouse macrophage NOS cDNA clone and crossspecies Northern blot hybridization, we demonstrated that LPS, TNF, IL-1, and IFN-y acted synergistically in vitro to induce the highest rat hepatocyte NOS mRNA levels. The mRNA levels correlated with NOS enzyme activity determined by culture supernatant NO- + NO- levels as well as cGMP release. The great induction of NOS mRNA within only a few hours suggests that the regulation of hepatocyte NOS expression is primarily transcriptional, as seen with macrophage NOS (13). Inhibition of NOS mRNA by actinomycin D is also consistent with this notion. That cycloheximide inhibits NOS mRNA induction suggests that a key polypeptide component of the cytokine signal transduction pathway, possibly including NOS transcriptional factors, is labile. It is unlikely that the inhibition by cycloheximide is simply due to cytotoxicity, as cycloheximide at 10 ,ug/ml does not abolish mRNA induction by cAMP in cultured rat hepatocytes (26). The increase in NOS mRNA from freshly isolated hepatocytes following C. parvum injection verifies that hepatocyte NOS is also induced in vivo during certain conditions of hepatic inflammation. The time delay of 4-6 hr required for cytokine and LPS induction of hepatocyte NOS activity is characteristic of the inducible macrophage NOS (27). However, the cytokine signals which up-regulate hepatocyte NOS differ quantitatively and qualitatively from those required to induce macrophage NOS. First, macrophage NOS activity has been reported to be maximally induced by two stimuli (27, 28), whereas both rat (19) and human (20) hepatocytes require a combination of four stimuli for the greatest induction. Second, the relative potency of the stimuli are cell-specific. Maximal macrophage NOS activity is typically reported following stimulation with LPS and IFN-y (27, 28). In contrast, in the current study in hepatocytes where NOS activity as well as mRNA levels were measured, we found that LPS + IFN-'y was the least effective stimulus in activating hepa-
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FIG. 6. In vivo vs. in vitro induction of hepatocyte NOS mRNA.
(Left) Northern blot analysis of NOS mRNA from freshly isolated hepatocytes of normal and C. parvum-injected rats. (Right) Northern blot analysis of hepatocytes exposed to CM for 6 hr in vitro.
526
Cell Biology: Geller et al.
tocyte NOS, achieving only 6-9%o of maximal measurable mRNA levels. Instead, LPS + TNF + IL-1 + IFN-y produced maximal hepatocyte NOS mRNA levels and nitrogen oxide synthesis. TNF + IFN-y and TNF + IL-1 were the most effective double signal combinations in hepatocytes, producing 35-45% of maximal mRNA levels. Numerous reports have documented synergistic actions between TNF and IFN-y (see ref. 29 for review) for activation of macrophage cytotoxicity and between TNF and IL-1 for induction of the hepatic acute-phase response. TNF has also been shown to directly stimulate NO' formation in cultured hepatocytes isolated from rats exposed to LPS (30). Whereas strong induction of NOS activity and marked increases in the mRNA were seen with cytokine and/or LPS combinations, increases in the mRNA were also seen when TNF, IL-1, or IFN-y was used alone. However, only TNF and, to a lesser extent, IL-1 increased NO- formation as indicated by NOj + NO- or cGMP release. The lack of NO+ NO- increase with IFN-y may be due to the sensitivity of our methods to detect NO- or the absence of NOS activity despite the presence of mRNA. These data suggest that as single agents, TNF and IL-1 are capable of stimulating low-level NOS activity in hepatocytes. The physiological significance of NO' biosynthesis in the liver is only beginning to be elucidated. In vitro hepatocyte NO' production has been associated with a decrease in hepatocyte total protein synthesis (18, 31) and mitochondrial aconitase activity (32) and an increase in cGMP synthesis and release (23). The cytostatic effect of macrophage NO' production against intracellular parasites (see ref. 33 for review) has also been shown in hepatocytes in vitro for inhibition of the pre-erythrocytic liver stages of malaria (34). Following the in vivo administration of LPS, NOS activity in the liver increased (35), and an endotoxin-induced NOS enzyme was recently purified (36). Chronic LPS infusion in vivo renders hepatocytes inducible for NO' production following singlecytokine stimulation (30). Taken together, these findings indicate that hepatic NOS is expressed in endotoxemia and sepsis and suggest a critical role for NO- in the liver as part of the host immune response. Other studies from our laboratory have looked into the function of NO- in the liver in vivo and have revealed that inhibition of NO' synthesis by NMA in a model of hepatic injury produced by C. parvum and LPS results in micro infarcts and increased liver damage mediated partially by oxygen radicals (17). This indicates that NO- is protective by preventing cell damage, organ ischemia, and infarction. From the results presented in the current paper it is clear that this induction of NOS in the liver is regulated by multiple signals often acting in synergy, and further studies are needed to define the precise mechanisms by which these signals activate the NOS gene. We thank Debra Williams for expert technical assistance and Sidney M. Morris, Ph.D., and David J. Tweardy, M.D., of the University of Pittsburgh for helpful discussions and suggestions. This work was supported by National Institutes of Health Grants GM44100 (T.R.B.) and GM37753 (R.L.S.). 1. Stuehr, D. J. & Marletta, M. A. (1985) Proc. Natl. Acad. Sci. USA 82, 7738-7742. 2. Hibbs, J. B., Jr., Taintor, R. R. & Vavrin, Z. (1987) Science 235, 473-476. 3. Palmer, R. M. J., Ferrige, A. G. & Moncada, S. (1987) Nature (London) 327, 524-526. 4. Ignarro, L. J., Buga, G. M., Wood, K. S., Byrns, R. E. & Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. USA 84, 92659269. 5. Busse, R. & Mulsch, A. (1990) FEBS Lett. 265, 133-136.
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