We recently reported an involvement of ANG II and the ANG II type 1 (AT1) receptor in the hepatic expression of IL-1β induced in dehydrated rats by LPS. Here, we first confirmed that ANG II and AT1 receptors contribute to the LPS-induced increase in the splenic concentration of IL-1β in dehydrated rats. We then investigated whether ANG II contributes to IL-1 production through a modulating effect on the activation of proinflammatory transcription factors (NF-κB and AP-1) that is induced in the dehydrated rat's liver and spleen by intravenous injection of LPS. Surprisingly, LPS markedly increased the hepatic activation of NF-κB, an effect that was significantly enhanced (rather than reduced) by pretreatment with an ANG-converting-enzyme (ACE) inhibitor or AT1-receptor antagonist. Furthermore, the same ACE inhibitor and AT1-receptor antagonist each increased the resting NF-κB activity in the liver and spleen, although they had no effect on the LPS-induced splenic expression of NF-κB. Both hepatic and splenic AP-1 expressions were enhanced by LPS. This response was significantly augmented by pretreatment with the AT1-receptor antagonist (but not with the ACE inhibitor) in the spleen, while in the liver, neither drug had any effect. These results suggest that the endogenous ANG II or AT1 receptor suppresses the activation of hepatic or splenic transcription factors in dehydrated rats given LPS. Our results seem not to support the idea that NF-κB and AP-1 play key roles in the ANG II-induced enhancement of the production of proinflammatory cytokines that is induced by LPS in dehydrated rats.
- electrophoretic mobility shift assay
it is widely accepted that when monocytes/macrophages are stimulated with LPS, they produce and release proinflammatory cytokines such as IL-1, which elicit several host-defense responses, including fever (4, 12). Furthermore, it is well known that an enhancement of fever occurs under dehydrated conditions. A few years ago, we found 1) that ANG II, the secretion of which increases under dehydrated conditions, contributes to the enhanced fever that is seen in dehydrated rats after an intravenous injection of LPS, but 2) that the fever induced by IL-1 does not involve an action of ANG II (24). More recently, we demonstrated an involvement of ANG II and the ANG II type 1 receptor (AT1 receptor) in the LPS-induced expression of IL-1 in the liver in dehydrated rats (15). Collectively, the above results make it seem likely that ANG II and the AT1 receptor play important roles in the LPS-induced production of proinflammatory cytokines, leading to the enhancement of LPS-induced fever that is seen under dehydrated conditions. However, at the present time, the mechanism underlying such ANG II-stimulated enhancement of the LPS-induced production of cytokines remains unknown.
For some time, LPS has been thought to activate the transcription factors NF-κB and activator protein-1 (AP-1) that regulate the gene expressions of many proinflammatory cytokines (1, 2, 7). In addition, it has been reported that ANG II activates NF-κB and AP-1 in monocytes (13, 19). Taken together, the above evidence makes it seem likely that under dehydrated conditions, ANG II mediates the LPS-induced activation of such transcription factors, leading to an increase in cytokine production.
To test this possibility, we examined the effects of an angiotensin-converting enzyme (ACE) inhibitor and those of an AT1-receptor antagonist on the LPS-induced activations of NF-κB and AP-1 in the liver and spleen of dehydrated rats, two organs representative of the reticuloendothelial system. The role of ANG II in the LPS-induced increase in the splenic concentration of IL-1β was also investigated, because no reports have been published on this matter in that organ.
MATERIALS AND METHODS
The animals used in this study were male Wistar rats weighing 270–290 g. They were housed in individual plastic cages [40 × 25 × 20 cm; (length × width × depth)] with wood-chip bedding in a room maintained at 26 ± 1°C, a temperature within the thermoneutral zone for rats. They experienced a 12:12-h light-dark photoperiod, lights coming on at 0700. All animals had ad libitum access to drink and standard laboratory rat chow. The protocols were reviewed and approved by the Committee on the Ethics of Animal Experiments in Tottori University Faculty of Medicine, and the experiments were carried out in accordance with the Guidelines for Animal Experiments at Tottori University Faculty of Medicine and the Federal Law (No. 221) and Notification (No. 6) of the Japanese Government.
This study comprised four experiments (experiments 1–4), all on freely moving rats. In experiments 1–3, all rats were dehydrated by deprivation of drinking water for 24 h before experimentation. Rats lost ∼8% of their total body weight as a result of this deprivation. In experiment 4, the rats were either dehydrated or euhydrated (see below). Each rat took part in only one experiment. Details of the experimental protocols are given below.
To permit intravenous injections, all rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a polyvinyl tube was inserted into the jugular vein so that its tip lay in the superior caval vein near the right atrium. The free end of the catheter was passed subcutaneously to the midscapular region, where it was exteriorized dorsally behind the neck. It was kept patent by flushing it every day with heparinized 0.9% saline (50 U/ml). This implantation was performed at least 3 days before the start of the experiment. A rat's body weight usually decreases a little (∼5 g) in the first day after such surgery. However, within 3 days, the weight starts to increase and reaches a level higher than that seen before the surgery. We excluded rats from the present data whose body weight did not recover in that way after the surgery. Furthermore, we have previously measured the resting ACTH level in the rat's plasma at 3 days after surgery of intravenous cannula implantation, and no increase in the level (which was about 25–30 pg/ml) was observed (25). Because proinflammatory stimuli such as LPS (23) or IL-1 (25) markedly increase the plasma ACTH, we believe that no significant inflammation was present at 3 days after the jugular catheter-implantation surgery.
For experiment 4 (see below), each rat was previously anesthetized with pentobarbital sodium (50 mg/kg ip) to allow a battery-operated transmitter (model TA10TA-F40) for the measurement of body temperature to be implanted intraperitoneally. This was done at least 7 days before the implantation of the intravenous cannula (see above) to permit measurement of body temperature using a biotelemetry system (Data Science, St. Paul, MN). The output of the transmitter was monitored by antennae mounted in a receiver board (model CTR86) placed under each animal's cage. The data were fed into a peripheral processor (matrix model BCM100) connected to a Sanyo MBC-17J AX computer (IBM compatible).
All rats were handled for 15 min each day for at least 5 days to get them accustomed to the experimenters.
The LPS used in this study, which was derived from Salmonella typhosa endotoxin (Sigma, St. Louis, MO), was dissolved in sterile saline. Lisinopril (Sigma) was also dissolved in sterile saline. Losartan, dissolved in sterile saline for injections, was a kind gift from Merck (Whitehouse Station, NJ). We examined the effects of lisinopril (20 mg/kg iv) or losartan (30 mg/kg iv) on LPS (5 μg/kg iv)-induced changes in this study. There are previous reports showing that an even smaller dose (5 or 10 mg/kg) of lisinopril is effective at inhibiting ACE (8, 9). Furthermore, less than 30 mg/kg of losartan is sufficient to inhibit ANG II-induced responses such as increases in water intake (18) and arterial blood pressure in rats (22). Therefore, we believe that the doses of lisinopril and losartan used in this study were sufficient to inhibit ACE and ANG II-mediated effects.
We investigated the effect of an intravenous injection of the ACE inhibitor lisinopril (20 mg/kg) or the AT1-receptor antagonist losartan (30 mg/kg) on the LPS (5 μg/kg iv)-induced changes in the splenic concentration of IL-1β. Rats were divided into two groups.
An injection of LPS (5 μg/kg iv) was given 30 min after an intravenous injection of either lisinopril (20 mg/kg; Lisinopril+LPS group) or saline (1 ml/kg; Saline+LPS group). The control rats received an intravenous injection of saline (vehicle for LPS) 30 min after intravenous saline (vehicle for lisinopril) (Saline+saline group).
Rats were given losartan (30 mg/kg iv; Losartan+LPS group) or saline (1 ml/kg iv; Saline+LPS group) 30 min before the LPS injection (5 μg/kg iv). The control rats received an intravenous injection of saline (vehicle for LPS) 30 min after intravenous saline (vehicle for losartan) (Saline+saline group).
To minimize the influence of the rat's own circadian rhythm, the injections in each group were always performed between 1100 and 1200.
Animals of both groups were killed by CO2 stunning followed by decapitation 2 h after their second injection (LPS or saline). The spleen was quickly removed, frozen, and powdered in liquid nitrogen.
The splenic concentration of IL-1β was measured by ELISA. In brief, each powdered tissue, immersed in Iscove's culture medium containing a cocktail protease inhibitor (Sigma), was mechanically homogenized on ice, using a postmounted laboratory homogenizer (Omni International, Warrenton, VA). Homogenized samples were centrifuged at 10,000 rpm for 10 min at 4°C. Supernatants were then transferred into a fresh test-tube and stored at −85°C until needed for measurement of IL-1β and total protein content. The IL-1β content was measured using a commercial ELISA kit (TFB, Tokyo, Japan) with a lower detection limit of 3 pg/ml. The total protein content was determined using a Bio-Rad protein-assay kit. The tissue concentration of IL-1β is expressed as the cytokine content/100 μg protein.
An intravenous injection of LPS (5 μg/kg) or saline (1 ml/kg) was given to allow us to observe the time-related changes in the activities of transcription factors (NF-κB and AP-1) in the liver or spleen. Animals were killed by CO2 stunning followed by decapitation either 15 min, 30 min, or 2 h after the injection. The liver and spleen were quickly removed, frozen, and powdered in liquid nitrogen. The activities of NF-κB and AP-1 were measured by means of an electrophoretic mobility shift assay (EMSA), following nuclear extraction (see below).
In brief, each powdered tissue, immersed in buffer 1 [50 mM Tris (pH 7.4), 1 mM Na-ortho-vanadate, cocktail protease inhibitor (Sigma)], was homogenized on ice. After the homogenate had been kept on ice for 30 min, it was centrifuged at 7,000 g for 10 min at 4°C. The pellet was suspended in Buffer 2 [20 mM HEPES (pH 7.9), 350 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 20% glycerol, 1% NP-40] on ice for 30 min. After centrifugation at 13,000 g for 10 min at 4°C, the supernatant liquid, containing nuclear protein, was collected. After the protein concentration of the nuclear extract had been determined using a Bio-Rad protein-assay kit, the samples were diluted with Buffer 2, so as to adjust their protein concentration to 6 μg/μl (liver) or 5 μg/μl (spleen). They were then stored at −80°C until needed.
Probes for NF-κB and AP-1 were synthesized as double-stranded oligomers: 5′ -AGTTGAGGGGACTTTCCCAGGC-3 and 5′ -CGCTTGATGACTCAGCCGGAA-3′, respectively. These probes were end-labeled with T4 polynucleotide kinase and [γ-32P]ATP (ICN).
Either 30 μg (liver) or 20 μg (spleen) of nuclear extract was preincubated on ice for 30 min with 1 μg of poly (dI-dC) (Roche Diagnostics, Indianapolis, IN) and 1 μg bovine serum albumin (New England BioLabs, Ippswich, MA) in 14 μl of gel shift buffer [for NF-κB (in mM): 25 HEPES (pH 7.9), 40 KCL, 5 MgCl2, 0.1 EDTA, 1 DTT, 0.5 PMSF, 5 ZnSO4, 20% glycerol: for AP-1 (in mM), 25 HEPES (pH 7.9), 80 KCl, 5 MgCl2, 0.1 EDTA, 1 DTT, 0.5 PMSF, 5 ZnSO4, and 7.5% glycerol]. The probe (10,000 cpm/sample) was then added to the medium, which was subsequently incubated for 30 min at a temperature appropriate for each probe (at 25°C for NF-κB, on ice for AP-1). Next, each sample was subjected to 5% PAGE in buffer [(in mM): 50 Tris, 380 glycine, and 2 EDTA (pH 8.2)]. Autoradiography was performed on the dried gels using Fuji Medical X-ray film (Fujifilm). For the EMSA supershift experiments, nuclear extracts were preincubated for 30 min at 25°C with 1 mg of polyclonal antibody against either p50-NF-κB or Jun-D (Santa Cruz Biotechnology, Santa Cruz, CA) before addition of the labeled probe. Competition assays were performed using a 100-fold excess of unlabeled probe. Quantification of the present EMSA was achieved using BAS2000 (Fuji Photo Film, Tokyo, Japan). The incubation time of the BAS was 24 h, and an area of 150–250 mm2 was measured for its photo-stimulated luminescence.
We investigated the effect of an intravenous injection of lisinopril (20 mg/kg) or losartan (30 mg/kg) on the activities of hepatic and splenic transcription factors (NF-κB and AP-1) at 30 min after an intravenous injection of LPS (5 μg/kg). We chose the 30-min time point because we felt it might be a time at which LPS is solely activating those transcription factors responsible for the genesis of IL-1β [indeed, it takes about 30 min for the transcription of IL-1β to take place (14)]. After that time, both LPS and the LPS-produced cytokines, such as IL-1β and IL-6, activate the transcription factors that contribute to the transcription of many additional genes (2). We wanted to know whether ANG II contributes to the activation of the transcription factors induced by LPS itself that may be involved in the production of IL-1β. The dose of lisinopril used in this study (20 mg/kg) was greater than that (10 mg/kg) previously used by us to show its effect on fever (24), but the same as that used by us to show an effect of ANG II on cytokines (15). As shown in the fever paper (24), at a dose of 10 mg/kg, lisinopril reduced the LPS-induced fever, although its effect seemed to be small. This is why we injected a higher dose of lisinopril in the IL-1β study (15). Furthermore, the purpose of the present study was to investigate whether ANG II contributes to IL-1 production through its modulating effect on the activation of proinflammatory transcription factors. Therefore, we used the higher dose of lisinopril (20 mg/kg) in this study.
The rats were divided into four groups (Saline+saline group, Saline+LPS group, Lisinopril+LPS group, and Losartan+LPS group). The timings of the injections and the doses of drugs were the same as those described for experiment 1. In addition, to examine the effect of each drug alone on the resting activities of the transcription factors, lisinopril or losartan was injected intravenously 30 min before a saline injection (Lisinopril+saline or Losartan+saline group).
The effect of dehydration on LPS (5 μg/kg iv)-induced fever was examined because our previous study (24) showing enhanced fever in dehydrated rats used a lower dose of LPS (2 μg/kg iv) than that used in the current study (5 μg/kg iv). The reason for this use of a higher dose of LPS was as follows. LPS obtained from Sigma (St. Louis, MO) had to be used in this study because the LPS from Difco Laboratories used in our previous study (24) is no longer available (since Difco Laboratories have stopped production of LPS). As reported in our previous paper (15), the LPS from Difco produced a significantly greater IL-1β response in the liver than that from Sigma. Because LPS activities would seem to differ, depending on the source, we used a higher dose of LPS (from Sigma) in this study.
Each dehydrated or euhydrated rat was gently picked up, and its transmitter was switched on with a magnet at 18 h before the start of the experiment. The body temperature was then allowed to stabilize at an ambient temperature of 26 ± 1°C before any injections. LPS was given intravenously to each animal over a period of 30 s. To minimize the influence of the rat's own circadian rhythm, LPS was always given between 1100 and 1200. We measured the changes in body temperature due to LPS for 7 h after the injection.
All results are expressed as means ± SE. IL-1β and EMSA data (experiments 1–3) were analyzed for statistical significance by means of a one-way ANOVA, followed by Fisher's paired least significant difference (PLSD) test (post hoc test) (Macintosh, StatView 4.0). Body-temperature data (experiment 4) were analyzed for statistical significance by means of a repeated-measures ANOVA, followed by Fisher's PLSD test (post hoc test) to assess the overall effect (Macintosh, StatView 4.0). This analysis was performed on data collected from the time of drug injection onward (i.e., from time 0 to 420 min). Details of the results of this analysis are given in the legend to Fig. 7. Differences were considered significant at P < 0.05.
Effect of Intravenous Treatment with Lisinopril or Losartan on Splenic Concentration of IL-1β in Dehydrated Rats Given Intravenous Injection of LPS (Experiment 1)
As shown in Table 1, the splenic concentration of IL-1β was significantly increased at 2 h after an intravenous injection of LPS (5 μg/kg, Saline+LPS group vs. Saline+saline group). In rats pretreated with lisinopril (20 mg/kg, Lisinopril+LPS group) or losartan (30 mg/kg, Losartan+LPS group), this LPS-induced IL-1β response was significantly reduced. The Saline+saline and Saline+LPS groups listed in Table 1 differ between the lisinopril and losartan experiments.
LPS-Induced Activation of NF-κB in Liver and Spleen of Dehydrated Rats (Experiment 2)
Figure 1 shows NF-κB activation in the liver and spleen in saline- and LPS-injected dehydrated rats. A time-related activation of NF-κB was observed in the liver (Fig. 1A) and spleen (Fig. 1B) after an intravenous injection of LPS (5 μg/kg). As shown in Fig. 1, C and D, the addition of cold oligo probes for NF-κB (competitor), but not for AP-1, reduced the shifted bands. Furthermore, the use of an antibody specific for the p50 subunit of NF-κB, but not normal rabbit serum (NRS), caused a shift of the NF-κB band.
The saline data presented in Fig. 1, A and B were obtained at 15 min postinjection. There were no significant differences among the NF-κB activity levels at 15, 30, and 120 min after saline injection in either the liver (fold increase at 15 min, 1.00 ± 0.07; at 30 min, 0.68 ± 0.13; at 120 min, 0.77 ± 0.12) or spleen (fold increase at 15 min, 1.00 ± 0.07; at 30 min, 0.76 ± 0.13; at 120 min, 0.94 ± 0.13).
Effects of Lisinopril and Losartan on LPS-Induced Activation of NF-κB in Liver of Dehydrated Rats (Experiment 3)
Figure 2, A and B shows that LPS (5 μg/kg iv) had induced an activation of hepatic NF-κB at 30 min after the LPS injection (Saline+LPS group vs. Saline+saline group). Surprisingly, this effect was significantly enhanced (rather than reduced) by intravenous treatment with either lisinopril (Lisinopril+LPS group, Fig. 2A) or losartan (Losartan+LPS group, Fig. 2B).
As depicted in Fig. 2, C and D, lisinopril (Lisinopril+saline group) and losartan (Losartan+saline group) each induced NF-κB activation.
Effects of Lisinopril and Losartan on LPS-Induced Activation of NF-κB in Spleen of Dehydrated Rats (Experiment 3)
Figure 3 shows that neither lisinopril (Lisinopril+LPS group, Fig. 3A) nor losartan (Losartan+LPS group, Fig. 3B) altered the LPS (5 μg/kg iv)-induced activation of splenic NF-κB (Saline+LPS group) in dehydrated rats. In contrast, the resting NF-κB activity was significantly increased in both the Lisinopril+saline (Fig. 3C) and Losartan+saline (Fig. 3D) groups.
LPS-Induced Activation of AP-1 in Liver and Spleen of Dehydrated Rats (Experiment 2)
Injection of LPS (5 μg/kg iv) induced time-related increases in AP-1 activity in the liver (Fig. 4A) and spleen (Fig. 4B) in dehydrated rats. As shown in Fig. 4C and D, the addition of cold oligo probes for AP-1 (competitor), but not for NF-κB, reduced the shifted bands. Furthermore, the use of an antibody specific for the Jun-D subunit of AP-1, but not NRS, caused a shift of the AP-1 band.
The saline data presented in Fig. 4A and B were obtained at 15 min postinjection. There were no significant differences among the AP-1 activity levels at 15, 30, and 120 min after saline injection in either the liver (fold increase at 15 min, 1.00 ± 0.08; at 30 min, 1.01 ± 0.04; and at 120 min, 1.07 ± 0.11) or spleen (fold increase at 15 min, 1.00 ± 0.08; at 30 min, 0.84 ± 0.04; and at 120 min, 1.38 ± 0.24).
Effects of Lisinopril and Losartan on LPS-Induced Activation of AP-1 in Liver of Dehydrated Rats (Experiment 3)
At 30 min after an intravenous injection of LPS (5 μg/kg), there were no differences in the LPS-induced increases in hepatic AP-1 activity between the Saline+LPS and Lisinopril+LPS groups (Fig. 5A), or between the Saline+LPS and Losartan+LPS groups (Fig. 5B). Furthermore, neither lisinopril (Fig. 5C) nor losartan (Fig. 5D) had any effect on the resting AP-1 activity.
Effects of Lisinopril and Losartan on LPS-Induced Activation of AP-1 in Spleen of Dehydrated Rats (Experiment 3)
As depicted in Fig. 6, the LPS (5 μg/kg iv)-induced activation of splenic AP-1 (Saline+LPS group) was significantly enhanced (rather than reduced) by intravenous pretreatment with losartan (Losartan+LPS group, Fig. 6B), although lisinopril (Lisinopril+LPS group, Fig. 6A) had no such effect. The intravenous injection of lisinopril (Fig. 6C) or losartan (Fig. 6D) had no significant effect on the resting level of splenic AP-1 activity in dehydrated rats (Lisinopril+saline and Losartan+saline groups).
Effects of Dehydration on LPS-Induced Fever in Rats (Experiment 4)
As shown in Fig. 7, the biphasic fever (latency, about 90 min) induced in euhydrated rats by an intravenous injection of LPS (5 μg/kg) was significantly enhanced in dehydrated rats, suggesting that at this dose of LPS, dehydration-induced changes (such as an increase in ANG II) contribute to the induction of LPS-induced fever in rats (24).
In this study, we examined the effect of lisinopril (ACE inhibitor) and that of losartan (AT1-receptor antagonist) on the LPS-induced activations of NF-κB and AP-1 in dehydrated rats. We did this to test the hypothesis that ANG II may activate these transcription factors, leading to an enhancement of the LPS-induced production of IL-1β. The present results revealed time-related activations of NF-κB and AP-1 both in the liver and in the spleen after an intravenous injection of LPS. Surprisingly, however, both the ACE inhibitor and the AT1-receptor antagonist enhanced (rather than reduced) the LPS-induced NF-κB and AP-1 responses, indicating that the endogenous ANG II and AT1 receptor may inhibit these transcription factors. On the other hand, the same drugs (lisinopril and losartan) reduced the LPS-induced increase in the IL-1β concentration in the spleen, as we have previously shown them to do in the liver (15). Because NF-κB and AP-1 are important for the expression of the IL-1β gene (1, 2, 20), our results do not seem to support the idea that NF-κB and AP-1 play key roles in the ANG II-induced enhancement of the production of proinflammatory cytokines that is induced by LPS in dehydrated rats.
However, previous studies have shown an inhibition by an ACE inhibitor or an AT1-receptor antagonist of the activation of NF-κB and/or AP-1 that is induced in the heart by myocardial infarction (26) and in the kidney by ureteral obstruction (16). Furthermore, exogenously administered ANG II activates NF-κB and AP-1 in the liver in vivo (3) and in cultured cells (such as monocytes) in vitro (13, 19). These previous results indicate that ANG II can indeed activate these transcription factors. How then can we explain the discrepancies between our results and the previous ones? First, we tested the effects of the drugs at only one time point (namely, at 30 min after the LPS injection). We chose this time point because the transcription of IL-1β begins at around 30 min (14), and we therefore thought that any activation of transcription factors after this time might be attributable not only to LPS, but also to already produced multiple cytokines such as IL-1, IL-6, and TNF. Indeed, activation of transcription factors at 2 h is not responsible for the IL-1β production observed at 2 h after an LPS injection, a production that is inhibited by the drugs mentioned above (see Table 1 and Ref. 15). Because the possibility cannot be excluded that at other time-points, lisinopril and losartan might inhibit the activations of transcription factors that are induced by both LPS and cytokines, we performed an additional experiment in which the effects of lisinopril and losartan on the activities of transcription factors were investigated at 120 min after the injection of LPS. The results showed that at this time point, there were no differences in the LPS-induced activations of the hepatic and splenic transcription factors between the Saline+LPS and Lisinopril+LPS or Losartan+LPS groups (see Table 2). These results suggest that at 120 min, ANG II does not play an important role in the LPS-induced activation of transcription factors, although we must keep in mind the possibility that there could still be different time points at which the drugs do inhibit the activation of transcription factors. Second, we used only one dose of LPS (5 μg/kg) and the route of injection may have been important. Third, we did not verify the effects of lisinopril and losartan on ANG II levels, and this omission needs to be remedied in the near future. Finally, it should be noted that there is one previous report showing an enhancement by an ACE inhibitor of the activation of NF-κB and AP-1 that occurs in the noninfarcted part of the myocardium in rats with a myocardialinfarction (5). On this basis, it seems unlikely that endogenous ANG II acts solely as an activator of these transcription factors under all conditions.
In the present study, both the ACE inhibitor and the AT1-receptor antagonist enhanced the LPS-induced NF-κB response in the liver. Furthermore, both drugs increased the resting level of NF-κB activity in the liver, suggesting that endogenous ANG II inhibits the hepatic resting NF-κB activity, an effect that might, in part, be responsible for the inhibition by ANG II of the LPS-induced NF-κB response. Because ANG II activates NF-κB in monocytes (13, 19), one possibility is that NF-κB activation in Kupffer cells, a kind of macrophage/monocyte, is actually inhibited by these drugs, but that they also enhance the LPS-induced NF-κB response in other cells and that this overrides the effect on the Kupffer cells. In other words, in non-Kupffer cells, such as hepatocytes, ANG II might inhibit the activation of NF-κB. In fact, it has been reported that ANG II, through an activation of the AT1 receptor, inhibits the IL-1-induced activation of NF-κB in rat-cultured vascular smooth muscle cells, leading to inhibition of inducible nitric oxide synthase (iNOS) expression (because NF-κB stimulates the transcription of the iNOS gene) (10). Because iNOS is induced by LPS in hepatocytes (6), it is possible that ANG II inhibits the LPS-induced NF-κB response in those cells, which, in turn, would reduce the production of iNOS. Furthermore, it is conceivable that there are other unknown NF-κB-mediated proteins whose transcription (like that of iNOS) is inhibited by ANG II in hepatocytes. These possibilities need to be investigated using hepatocyte cell-culture in the not-too-distant future. The present results show that in the spleen, the LPS-induced activation of AP-1 was enhanced by an AT1-receptor antagonist, but not by an ACE inhibitor, suggesting that ANG II does not alter AP-1 activity in this organ. However, it is possible that the AT1 receptor might inhibit and the AT2 receptor facilitate AP-1 activity, because in some circumstances, AT1 and AT2 receptors exert opposite effects on ANG II-induced responses, one example being vasoconstriction (11, 21). The contribution made by ANG II receptors to the present response may be an another example. However, the possible inhibition by the AT1 receptor and facilitation by the AT2 receptor of the LPS-induced AP-1 response in the spleen, and its underlying mechanism, must await future research. In the present study, losartan itself tended to increase the resting AP-1 activity in the spleen (see Fig. 6D), although the effect did not reach significance. Therefore, we cannot exclude the possibility that losartan induced a slight activation of splenic AP-1, contributing in part to the facilitatory effect of this inhibitor on AP-1 activity in LPS-injected rats. At any rate, the above findings are different from those obtained for the liver. This may be directly related to differences in blood flow between these two organs and/or to the liver being an organ that is important for LPS clearance.
In this study, neither lisinopril nor losartan exerted any significant effect on the LPS-induced activation of splenic NF-κB, even though each drug by itself enhanced the splenic NF-κB activity. At the present time, although we cannot give a clear explanation for this discrepancy, one possibility occurs to us. That is, LPS may induce NF-κB activation to such a degree that the drugs were unable to increase it any further. Indeed, the NF-κB activity was increased 2.5-fold in the spleen of the LPS-injected rats (see Fig. 3, A and B). This raised level was probably very high, because the level achieved by the 2.7-fold increase seen in the spleen of LPS-injected rats at 120 min postinjection appears dense in the gels (as compared with that achieved after the ninefold increase in the liver) (see Fig. 1, A and B). In fact, when compared in the same gels, LPS induced a significant increase in splenic NF-κB activity at 120 min postinjection that elevated this activity to a level significantly greater than that achieved by hepatic NF-κB (unpublished observation). Therefore, there might be a ceiling effect of LPS on NF-κB activity.
In summary, the present results represent the first in vivo evidence that endogenous ANG II and AT1 receptors may inhibit the activation of transcription factors that is induced in the liver and spleen of the dehydrated rat by an intravenous injection of LPS. To our knowledge, the role of endogenous ANG II in LPS-induced NF-κB and AP-1 responses has never been examined in vivo, although Napoleone et al. (17) showed an inhibition by an ACE inhibitor of LPS-induced c-Rel/p65 NF-κB activation in monocytes in vitro. Therefore, we should not exclude the possibility that ANG II might activate transcription factors in one cell type but inhibit them in others. The resultant activities of such factors in a whole organ might therefore depend on the overall effect of ANG II, as it would act as both an activator and an inhibitor.
This work was partly supported by the Ministry of Education, Science and Culture with a Grant-in-Aid for Scientific Research (C15590209).
We are grateful to Dr. Robert J. Timms for his critical reading of the English manuscript. We thank Dupont Merck for the kind supply of losartan.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society