Rutgers University and University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway
Correspondence: Debra L. Laskin, Rutgers University, 160 Frelinghuysen Rd., Piscataway, NJ 08854. E-mail: laskin{at}eohsi.rutgers.edu
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(IFN-) had no major effect on COX-2, NOSII expression increased. Induction of acute endotoxemia resulted in a rapid and transient increase in constitutive COX-2 expression and prostaglandin E2 (PGE2) production by liver macrophages as well as NOSII expression and nitric oxide release. Cells from endotoxin-treated rats were also sensitized to generate more nitric oxide and express increased NOSII in response to LPS and IFN-. Inhibition of NOSII with aminoguanidine reduced COX-2 mRNA and protein expression as well as PGE2 production by activated macrophages from endotoxemic, but not control animals. In contrast, SC236, a specific COX-2 inhibitor, had no effect on NOSII mRNA or protein levels or on nitric oxide production by hepatic macrophages, even after endotoxin administration. These data suggest that activation of COX-2 may be important in the pathophysiological response of hepatic macrophages to endotoxin. Moreover, nitric oxide is involved in regulating COX-2 in activated liver macrophages during acute endotoxemia.
Key Words: PGE2 • NOSII • liver • Kupffer cells
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(IFN-) was obtained from Gibco-BRL (Grand Island, NY). Mouse monoclonal anti-NOSII antibody (clone N-39120) was from Transduction Laboratories (Lexington, KY). Goat polyclonal anti-COX-2 antibody (clone M-19), horseradish peroxidase (HRP)-conjugated anti-mouse, and anti-goat immunoglobulin G (IgG) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). SC236 {4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzynesulfonamide} was generously provided by Monsanto (St. Louis, MO).
Animals and treatments
Female-specific, pathogen-free Sprague-Dawley rats (200–225 g, 6–8 weeks of age) were obtained from Taconic (Germantown, NY). Animals were maintained on food and water ad libitum and housed individually in microisolator cages. To induce acute endotoxemia, rats were treated with 5 mg/kg LPS (intravenously) in phosphate-buffered saline (PBS). Control animals received 100 µl PBS.
Macrophage isolation
Cells were isolated from the livers as previously described with some modifications [27
, 28
]. Briefly, the livers were perfused in situ with Ca2+/Mg2+-free Hanks’ balanced salt solution containing 0.5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid and 25 mM HEPES followed by Leibovitz L-15 medium containing 100 U/ml collagenase type IV. Livers were then extracted, weighed, and combed, and the resulting cell suspension was filtered through 60 µm nylon mesh. Hepatocytes were separated from the nonparenchymal cells by three successive centrifugations (50 g, 2 min). Nonparenchymal cells were recovered by centrifugation of the supernatant at 300 g for 5 min. These cells were then purified on a Beckman J-6 elutriator (Beckman Instruments Inc., Fullerton, CA) equipped with a centrifugal elutriation rotor set to a pump speed of 12 ml/min and a rotor speed of 2500 rpm. Macrophages were collected at 44 ml/min. Cells were enriched by differential centrifugation on an 18% metrizamide gradient, were identified morphologically and by peroxidase staining, and were 80–85% pure.
Measurement of nitric oxide production
Cells were plated into 6-well dishes (2x106 cells/well) in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% penicillin (100 IU/ml)-streptomycin (100 µg/ml), and 0.05 IU/ml porcine pancreas insulin (complete DMEM). After 24 h in culture, the cells were washed and refed with DMEM with and without aminoguanidine, SC236, LPS, and/or IFN-. Nitric oxide production was quantified 24 h later by the accumulation of nitrite in the culture medium using the Greiss reaction with sodium nitrite as a standard [29
]. Absorbance was determined spectrophotometrically at 540 nm. For nitrate determinations, samples were treated with nitrate reductase and reduced nicotinamide adenine dinucleotide phosphate for 30 min prior to analysis using a nitrate/nitrite assay kit (Boehringer Mannheim, Indianapolis, IN). We found that in medium from cells cultured for 24 h with LPS and IFN-, the ratio of nitrate:nitrite was 1.0:1.2 for hepatic macrophages. This ratio did not change in cells isolated from rats treated with endotoxin.
Measurement of prostaglandin E2 (PGE2) production
Cells were plated into 12-well dishes (1x106 cells/well) in complete DMEM. After 2 h in culture, the cells were washed and re-fed with DMEM with and without aminoguanidine, SC236, LPS, and/or IFN-. PGE2 in culture supernatants was quantified 24 h later using a commercial enzyme-linked immunosorbent assay kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Western blot analysis
Cells were plated into 6-well dishes (2x106 cells/well) in complete DMEM. After overnight incubation at 37°C, the cells were washed and incubated for 24 h in DMEM with and without aminoguanidine, SC236, LPS, and/or IFN-. The cells were then washed with PBS and lysed in buffer containing 10 mM Tris-HCl and 1% sodium dodecyl sulfate (SDS), pH 7.4. Protein concentrations were determined using the Pierce bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Cellular proteins (5 µg/lane) were fractionated on 7.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated with mouse monoclonal anti-NOSII antibody (1:1000) or goat polyclonal anti-COX-2 antibody (1:1000) for 1 h, followed by HRP-conjugated goat anti-mouse (1:2000) or rabbit anti-goat IgG (1:10,000) for 1 h, respectively. Antibody binding was visualized on film using enhanced chemiluminescence Western blotting reagents (Amersham Life Sciences, Arlington Heights, IL).
Relative reverse transcription-polymerase chain reaction (RT-PCR)
Cells were plated into 12-well dishes (2x106 cells/well) in complete DMEM. After 2 h at 37°C, the cells were washed and re-fed with DMEM containing LPS and aminoguanidine or medium control. The cells were then cultured for 18 h and washed with PBS, and total RNA was extracted using the RNeasy mini-kit (Qiagen, Valencia, CA). For first strand synthesis of liver cDNA, RNA (0.5 µg) in 9 µl water was denatured at 65°C for 4 min, cooled rapidly on ice, and then resuspended in a 20 µl final volume consisting of 50 mM Tris-HCl, pH 8.3, 40 mM KCl, 6 mM MgCl2, 0.1 mg/ml bovine serum albumin, 1 mM each deoxyribonucleotide triphosphate (dNTP), 20 mM random hexamers, and 500 U Superscript II RNase H- RT (Gibco-BRL, Gaithersburg, MD). After 1 h at 37°C, RNase H- (4 U) was added, and the samples were incubated at 37°C for an additional 20 min.
Prior to relative RT-PCR, the linear range of amplification for NOSII and COX-2 mRNA was determined for each treatment sample using mouse NOSII and COX-2 primer pairs (Ambion, Inc., Austin, TX) with the macrophage cDNA samples as templates, following the manufacturer’s directions. The primers used were: NOSII, sense 5'-AACTACTGCTGGTGGTGACA and antisense 5'-TTCGGACATCAAAGGTCTCA; COX-2, sense 5'-CATTCTTTGCCCAGCACTTCAC and antisense 5'-GACCAGGCACCAGACCAAAGAC. Nineteen cycles fell within the linear range for each control and treated sample (Fig. 1
). The 18S primer:competimer (Ambion, Inc.) ratio was then adjusted to produce an amplification signal comparable with the COX-2 signal. An 18S rRNA primer:competimer ratio of 1:9 was found to be appropriate to normalize each NOSII and COX-2 amplification signal. Macrophage cDNAs were then amplified in 20 µl buffer containing 1 µl cDNA (representing 0.9 µg total RNA), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 5 µM each NOSII and COX-2 primers, 1:9 ratio of 18S primers, 0.1 mM each deoxynucleotide triphosphate, 2.0 µl 
-32P deoxycytosine triphosphate (10 mCi/ml; >3000 Ci per mmol), and 0.025 units Amplitaq (PerkinElmer, Inc., Norwalk, CT). Amplification was initiated by 1 min denaturation at 94°C, followed by 19 cycles at 94°C for 15 s, 56°C for 25 s, and 72°C for 90 s.
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Figure 1. NOSII and COX-2 mRNA expression in hepatic macrophages. Cells isolated from control (CTL) animals or 24 h after endotoxin administration (ETX) were cultured with or without LPS (100 ng/ml) and analyzed for NOSII and COX-2 mRNA expression by semiquantitative RT-PCR. Left panel: the amount of radioactivity per band for each cycle. Triangles, cells from control animals; squares, cells from ETX-treated animals. Open symbols, unstimulated cells; closed symbols, LPS-stimulated cells. Right panel, Samples from unstimulated (-) or LPS-stimulated (+) cells, run for 19 cycles, were separated on 5% polyacrylamide gels.
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Statistics
Unless otherwise stated, all experiments used three to five animals per treatment or time point and were repeated two to three times. Data were analyzed using the analysis of variance (ANOVA) and Fisher LSD test. Results were considered statistically significant at P 
0.05.
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increased expression of the enzyme (Fig. 1
and not shown). Hepatic macrophages from control animals were also found to express COX-2 protein (Fig. 2
). As observed with mRNA expression, protein expression was increased after the cells were treated with LPS or LPS plus IFN- (Fig. 2
and not shown). Induction of acute endotoxemia resulted in a transient increase in COX-2 protein in the cells, which was evident within 1.5 h and maintained for 6 h. Subsequently, protein levels declined and by 24 h, were below control levels. At this time COX-2 mRNA levels were also below control (Fig. 1) . The early increase in COX-2 expression in macrophages after endotoxin administration was correlated with a rapid and transient increase in PGE2 production by the cells, which reached a maximum 1.5–3 h post-treatment (Fig. 2)
. PGE2 production, like COX-2 expression, declined with time after endotoxin administration to the animals. Treatment of cells from endotoxemic rats with LPS or LPS plus IFN- had no major effect on COX-2 mRNA or protein expression or on PGE2 production.
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Figure 2. Effects of acute endotoxemia on COX-2 protein expression and PGE2 production. Macrophages isolated from control (CTL) animals or 1.5–48 h after endotoxin administration (ETX) were cultured with (stimulated) or without (unstimulated) LPS (100 ng/ml) and IFN- (50 U/ml). After 24 h, cells and culture supernatants were collected and analyzed for COX-2 expression (upper panel) and PGE2 content (lower panel), respectively. One representative Western blot of five separate experiments is shown. PGE2 values represent the mean ± SEM from five different experiments.
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, caused a prolonged increase in NOSII expression by macrophages from endotoxemic rats (Fig. 3
and other data not shown). This was correlated with increased nitric oxide production by the cells. Nitric oxide production was also increased in unstimulated cells after endotoxin administration. As observed with NOSII expression, these effects were transient.
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Figure 3. Effects of acute endotoxemia on NOSII protein expression and nitric oxide production. Macrophages isolated from control (CTL) rats or 1.5–48 h after endotoxin administration (ETX) were cultured with (stimulated) or without (unstimulated) LPS (100 ng/ml) and IFN- (50 U/ml). After 24 h, cells and culture supernatants were collected and analyzed for NOSII expression (upper panel) and nitrite content (lower panel), respectively. One representative blot from six separate experiments is shown. Nitrite values represent the mean ± SEM from six different experiments. *, Significantly different (ANOVA, P0.05) from stimulated cells from control animals. **, Significantly different (ANOVA, P0.05) from unstimulated cells from control animals.
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Figure 4. Effects of NOSII and COX-2 inhibitors on enzyme expression and mediator production. Macrophages isolated from control rats (open bars) or 24 h after endotoxin administration (ETX, shaded bars) were treated with LPS (100 ng/ml) and increasing concentrations of the NOSII inhibitor, aminoguanidine (AG; left panels) or the COX-2 inhibitor, SC236 (right panels). After 24 h, cells and culture supernatants were collected and analyzed for NOSII and COX-2 expression (upper panels) and nitrite and PGE2 content (lower panels), respectively. One representative Western blot from three to nine separate experiments is shown. Nitrite and PGE2 values represent the mean ± SEM from three to nine different experiments. *, Significantly different (ANOVA, P0.05) from LPS.
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Figure 5. Effects of inhibition of NOSII on COX-2 expression and PGE2 production. Macrophages, isolated from control animals or 24 h after endotoxin administration (ETX), were incubated with medium control (Med) or LPS (100 ng/ml) and increasing concentrations of aminoguanidine (AG, mM). After 24 h, cells and culture supernatants were collected and analyzed for COX-2 protein (top panels) and mRNA expression (middle panels) and PGE2 content (lower panels). One representative Western blot and RT-PCR from two to four separate experiments is shown. PGE2 values represent the mean ± SEM from four different experiments. *, Significantly different (Fisher LSD, P0.05) from cells treated with LPS alone. **, Significantly different (Fisher LSD, P0.05) from cells treated with media.
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Figure 6. Effects of inhibition of COX-2 on NOSII protein expression and nitric oxide production. Macrophages, isolated from control rats or 24 h after endotoxin administration (ETX), were cultured with LPS (100 ng/ml) and increasing concentrations of SC236 (µM). After 24 h, cells and culture supernatants were collected and analyzed for NOSII expression (upper panels) and nitrite content (lower panels), respectively. One representative Western blot from nine separate experiments is shown. Nitrite values represent the mean ± SE from nine different experiments.
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Macrophages from control animals were found to express low levels of COX-2 and NOSII mRNA and protein and to produce small quantities of PGE2 and nitric oxide. Similar constitutive activity has been described previously in hepatic macrophages [43 44 45 46 47 ]. This is presumably a result of continuous exposure of these cells to gut-derived endotoxin. Acute endotoxemia resulted in a rapid increase in the activity of COX-2 and NOSII. However, by 48 h post-endotoxin treatment, production of PGE2 and nitric oxide by liver macrophages was at control levels. This suggests that these mediators play an important role in early inflammatory responses to endotoxin. Similar transient increases in PGE2 or nitric oxide release by hepatic macrophages have been described after ethanol [48 ], acetaminophen [49 ], or carbon tetrachloride [50 ] administration to rats.
LPS and/or IFN- have been shown to stimulate PGE2 release by hepatic macrophages [43
, 51
, 52
]. In contrast, we found that these mediators had no significant effect on this activity in liver macrophages, even after endotoxin administration. These differences may be a result of the prolonged culture times used in previous studies, which can alter macrophage functional responsiveness, and/or to higher doses of LPS and cytokines used to stimulate the cells. Prostaglandin production is also known to be limited by the availability of arachidonic acid [53
], and this may contribute to the lack of response of the macrophages in our model. In contrast, as observed previously [31
], LPS and IFN- induced liver macrophage nitric oxide production, a response that was increased after endotoxin administration. It is interesting that this was inversely correlated with time following treatment of the animals with endotoxin. Thus, cells isolated 24–48 h post-endotoxin treatment were significantly more responsive to LPS and IFN- than cells recovered after 3 h. Endotoxin has been shown to up-regulate cytokine receptor expression on macrophages [54
]. Increased sensitivity of macrophages to LPS and IFN- may be a result of increased expression of receptors for these inflammatory mediators on the cells. This possibility is consistent with the observation that 24–48 h were required for the response to be detected.
Inflammatory mediators released at sites of injury function to destroy invading pathogens and to initiate the process of wound repair. Coordinate regulation of mediator release is necessary for optimal host defense. In macrophages from control animals, expression of COX-2 mRNA and protein and PGE2 production were unaffected by the NOSII inhibitor, aminoguanidine. This suggests that under homeostatic conditions, pathways that regulate the expression and activity of these enzymes are independent. Stadler et al. [52 ] demonstrated that blocking nitric oxide synthesis with monomethyl L-arginine (L-NMMA) increased PGE2 production by resident Kupffer cells from untreated animals. Differences between our findings may be a result of differences in the method used to quantify PGE2 and/or the distinct actions of aminoguanidine and monomethyl L-arginine (L-NMMA). Thus, whereas aminoguanidine is relatively selective for NOSII, L-NMMA is also effective in blocking constitutive isoforms of NOS. L-NMMA, but not aminoguanidine, also blocks arginine uptake by macrophages [55 ]. In contrast to cells from control rats, macrophages from endotoxemic animals expressed reduced levels of COX-2 mRNA and protein and generated less PGE2 when NOSII activity was blocked with aminoguanidine. These findings support the idea that the effects of nitric oxide depend on the activation state of the cells [56 ]. Our results are in accord with previous studies demonstrating that nitric oxide positively regulates COX-2 in activated macrophages [7 , 8 , 57 58 59 60 ]. Prostaglandin biosynthesis has been reported to be significantly reduced in peritoneal macrophages from NOSII knockout mice after stimulation with inflammatory mediators [61 ]. The mechanism underlying this effect is unknown. Superoxide dismutase mimetics have been shown to decrease prostaglandin production by macrophages [62 ]. These data suggest that peroxynitrite, a potent oxidant formed from nitric oxide and superoxide anion, may be involved in this response [57 ].
Our studies also showed that NOSII activity in liver macrophages is independent of prostaglandin production. Thus, blocking COX-2 activity with SC236 had no effect on NOSII expression or nitric oxide production by macrophages even after endotoxin administration. Similar results have been observed using indomethacin or ibuprofen in control animals [63 ]. In murine J774 macrophages, low doses of prostaglandins exert a positive effect on LPS-stimulated NOSII expression, while high doses are inhibitory [64 ]. Selective COX-2 inhibitors have also been shown to reduce NOSII activity and expression in vivo [65 ]. These data suggest that the effects of prostaglandins on NOSII depend on the specific target cell population, as well as on the dose and timing of exposure.
The present studies demonstrate that hepatotoxic doses of endotoxin result in increased release of PGE2 and nitric oxide by hepatic macrophages. Moreover, during the pathogenesis of endotoxin-induced hepatic injury, nitric oxide is an important regulator of COX-2. That this is not observed in cells from control animals supports the idea that the activation state of the cells can influence regulatory pathways.
Received June 7, 2001; revised November 1, 2001; accepted December 15, 2001.
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