Introduction

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Sepsis is a systemic response to serious infection, and has a poor prognosis when it is associated with organ dysfunction, hypoperfusion, or hypotension (Parrillo, 1996). Gram-negative sepsis is initiated by exposure to the structural component of Gram-negative bacterial membrane, lipopolysaccharide (LPS), and induces the overproduction of host inflammatory cytokines, such as tumor necrosis factor- (TNF-) and interleukin-1 (IL-1; Hesse et al., 1988; Cannon et al., 1990), which in turn up-regulate the expression of inducible nitric-oxide synthase (iNOS; Förstermann et al., 1994; Nathan and Xie, 1994). The large amounts of cytokines (Tracy et al., 1987) and nitric oxide (NO; Julou-Schaeffer et al., 1990; Kilbourn et al., 1990; Petros et al., 1991) produced by iNOS are thought to contribute to LPS-induced hypotension, multiple organ system failure, and mortality.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, which are antihypercholesterolemic agents, reduce low-density lipoprotein cholesterol levels by blocking the mevalonate pathway and by increasing low-density lipoprotein receptor expression in the liver (Brown and Goldstein, 1986). HMG CoA reductase inhibitors have been reported to reduce the risk of coronary heart disease not only in patients with hypercholesterolemia (Shepherd et al., 1995) but also in individuals with average cholesterol levels (Downs et al., 1998). In addition, recent studies have shown that HMG CoA reductase inhibitors have various nonlipid effects, including antithrombotic properties (Mayer et al., 1992; Wada et al., 1993), antiproliferative effects (Munro et al., 1994; Corsini et al., 1996), induction of apoptosis (Jones et al., 1994; Tan et al., 1999), suppression of lymphocyte functions (Cutts and Bankhurst, 1989), and anti-inflammatory effects (Bustos et al., 1998; Pruefer et al., 1999). Pahan et al. (1997) reported that an HMG CoA reductase inhibitor could inhibit LPS-induced production of cytokines and NO in astrocytes, microglia, and macrophages in vitro. In this study, we therefore examined whether in vivo administration of an HMG CoA reductase inhibitor inhibits LPS-induced overproduction of host inflammatory mediators and prevents LPS-induced death.

	Materials and Methods

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Treatment of Mice. Male CD-1 mice (Charles River, Japan Inc., Kanagawa, Japan) were given a standard laboratory diet and water ad libitum and housed under controlled environmental conditions. They were 5 to 6 weeks of age at the start of experiments. After a minimum 7-day acclimation period, the mice were divided into two groups and given saline with or without cerivastatin sodium (20 mg/kg i.p., donated by Bayer Pharmaceuticals, Osaka, Japan) at 10 ml/kg 12 and 1 h before LPS administration under sterile conditions. Cerivastatin sodium is an entirely synthetic compound and produced sterilely. In addition, cerivastatin itself neither increased serum levels of TNF- nor affected the health of mice, such as their dietary intake or body weight (data not shown). LPS (Difco, Detroit, MI) was administered i.p. at a dose of 15 mg/kg. Survival of mice was monitored at intervals of 12 h for 7 days. All animal procedures were in accordance with the standards set forth in the Guidelines for the Care and Use of Laboratory Animals of the Takara-machi campus of Kanazawa University.

Serum TNF- and IL-1 Measurement. Mice were anesthetized with ether, and blood was collected by cardiac puncture. Serum levels of TNF- and IL-1 were determined by an enzyme-linked immunosorbent assay kit (Endogen, Woburn, MA) with a polyclonal rabbit anti-mouse TNF- or IL-1 antibody. Lower limits of quantification of TNF- and IL-1 were 10 and 3 pg/ml, respectively. Their intra- and interassay coefficients of variation were all <10%.

Serum Nitrite and Nitrate (NOx) Measurement. The serum samples were deprived of protein by filtration through molecular cut-off filters, microconcentrators 10 (Amicon, Beverly, MA), and assayed for NOx by the Griess method (Green et al., 1982) with a commercial kit (Dojindo, Kumamoto, Japan).

Statistical Analysis. Differences in serum TNF-, IL-1, and NOx levels between cerivastatin-pretreated and control mice were determined with the nonparametric Mann-Whitney test. The overall difference in survival rate of LPS-treated mice was determined by survival analysis, and P values were determined by the log-rank (Mantel-Cox) test. Values are presented as mean ± S.E., and significance of differences was assumed at P < .05. All calculations were performed with the computer program Statview, version 4.5, for Macintosh (Abacus Concepts, Berkeley, CA).

	Results

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Effects of Cerivastatin on Serum TNF- and IL-1 Levels in LPS-Treated Mice. Expression of inflammatory cytokines, such as TNF- and IL-1, is known to be induced by LPS in various types of cells in vitro (Pahan et al., 1997). Therefore, we first investigated the changes over time in serum levels of these mediators after LPS administration (15 mg/kg i.p.) in CD-1 mice. As shown in Fig. 1A, serum TNF- levels, which were not detectable before LPS injection, rose at 1 h, peaked at 2 h, and dropped to near basal levels by 4 h. Serum IL-1 levels also peaked at 2 h, but remained high longer than serum TNF- levels (Fig. 1B).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Time course of changes in serum levels of TNF- (A) and IL-1 (B) in LPS-treated mice. Male CD-1 mice at 5 to 6 weeks of age were given LPS (15 mg/kg i.p.), and blood samples were collected at 0, 1, 2, and 4 h after LPS injection. Serum levels of TNF- and IL-1 were determined as described under Materials and Methods. Values are mean ± S.E.; n = 3 to 4.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effects of cerivastatin on LPS-induced increase in serum levels of TNF- (A) and IL-1 (B) in mice. Male CD-1 mice at 5 to 6 weeks of age were given saline i.p. with () or without () cerivastatin (20 mg/kg) 12 and 1 h before LPS administration. LPS (15 mg/kg) was administered i.p. at 0 h. Blood samples were collected at 0 and 2 h after LPS injection. Values are mean ± S.E.; n = 3. P < .05.

Effect of Cerivastatin on Serum NOx Levels in LPS-Treated Mice. As reported previously (Tracey et al., 1995), CD-1 mice injected with 15 mg/kg LPS time dependently accumulated high serum levels of NOx, which reached about 16 times basal level at 8 h. Preadministration of cerivastatin (20 mg/kg i.p. 12 and 1 h before LPS injection) significantly reduced serum NOx level at 8 h by 44% without affecting basal NOx level (Fig. 3).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of cerivastatin on LPS-induced increase in serum levels of NOx in mice. Male CD-1 mice at 5 to 6 weeks of age were given saline i.p. with () or without () cerivastatin (20 mg/kg) 12 and 1 h before LPS administration. LPS (15 mg/kg) was administered i.p. at 0 h. Blood samples were collected at 0 and 8 h after LPS injection. Serum levels of NOx were determined as described under Materials and Methods. Values are mean ± S.E.; n = 3. P < .05.

Effect of Cerivastatin on Survival Rate of Mice Treated with LPS. Based on the above-mentioned findings, we examined whether cerivastatin could prevent LPS-induced death. Eleven of the 15 mice pretreated with saline alone (73.4%) died within 1 to 4 days after LPS injection (15 mg/kg; Fig. 4). Only 26.7% of mice pretreated with cerivastatin (20 mg/kg i.p. 12 and 1 h before LPS administration) died after LPS challenge. The overall difference in survival rate between groups with and without cerivastatin was significant (P = .016).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of cerivastatin on survival of LPS-treated mice. Male CD-1 mice at 5 to 6 weeks of age were given saline i.p. with (, n = 15) or without (, n = 15) cerivastatin (20 mg/kg) 12 and 1 h before LPS administration. LPS (15 mg/kg) was administered i.p. at day 0. Survival of the mice was monitored at intervals of 12 h for 7 days. The overall difference in survival rate between the groups with and without cerivastatin was significant (P = .016).

	Discussion

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HMG CoA reductase catalyzes the formation of mevalonate from acetyl-CoA, and regulates cholesterol biosynthesis. Mevalonate metabolites, particularly farnesyl pyrophosphate, are involved in post-translational modification of several important proteins such as cellular membrane G proteins and Ras proteins (Goldstein and Brown, 1990; Maltese, 1990). In addition, inhibitors of HMG CoA reductase have recently been shown to reduce LPS- and TNF--induced nuclear factor-B (NF-B) activation in mesangial cells, vascular smooth muscle cells, and mononuclear cells (Guijarro et al., 1996; Ortego et al., 1999). Furthermore, in rat macrophages, the HMG CoA reductase inhibitor lovastatin inhibited LPS-induced expression of cytokines and iNOS, which is thought to be mediated via NF-B activation (Pahan et al., 1997). Because these effects were not reversed by cholesterol and ubiquinone, which are end products of the mevalonate pathway, but were reversed by mevalonate and farnesyl pyrophosphate, the mevalonate pathway is thought to play an important role in controlling NF-B-mediated expression of cytokines and iNOS. In this study, we demonstrated for the first time in vivo that cerivastatin inhibits elevation of inflammatory cytokine and NO levels, although the molecular mechanisms underlying these effects remain to be investigated.

In concordance with inhibition of induction by LPS of cytokines and NO, cerivastatin actually improved survival of mice with LPS-induced sepsis. Sepsis frequently affects cholesterol metabolism, and LPS has been reported to provoke an increase in hepatic cholesterol synthesis in rodents that is accompanied by increase in HMG CoA reductase synthesis (Feingold et al., 1993). In addition, Memon et al. (1993) have shown that TNF- and IL-1 increase hepatic HMG CoA reductase activity and cholesterol synthesis in mice. These findings suggest that LPS-induced overproduction of cytokines and NO is strongly associated with activation of the mevalonate pathway, which can be blocked by HMG CoA reductase inhibitors. Whether cerivastatin prevents LPS-induced death via inhibition of the mevalonate pathway remains to be elucidated.

Serum lipoproteins are known to protect against endotoxin-induced death by binding and inactivating endotoxin (Harris et al., 1990). In this study, the administration of cerivastatin 12 and 1 h before measurement did not affect serum levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (data not shown). Therefore, cerivastatin does not seem to prevent LPS-induced sepsis by modifying serum lipoprotein profiles.

Severe sepsis unfortunately still has a high mortality rate because no single treatment has been found to be completely effective for it (Parrillo, 1996). Although overproduction of cytokines, such as TNF- and IL-1, is thought to play an important role in the development of sepsis (Hesse et al., 1988; Cannon et al., 1990), recent clinical trials of agents directed against TNF- or IL-1 have failed to improve survival of patients with sepsis (Fisher et al., 1993, 1994). In addition, overexpression of iNOS induced by LPS and/or cytokines in sepsis has been reported to cause hypotension and blood flow abnormality. However, it is still unclear whether iNOS-deficient mice are resistant to LPS-induced death (Laubach et al., 1995; MacMicking et al., 1995; Wei et al., 1995), and whether NOS inhibitors are effective in LPS-induced sepsis models (Kilbourn et al., 1990; Nava et al., 1992; Minnard et al., 1994). Because in this study cerivastatin inhibited not only the production of TNF- and IL-1 but also the production of NO, resulting in prevention of LPS-induced death, it might be very useful for prophylaxis of severe sepsis. However, the agent does not seem to be so effective against sepsis after the onset of severe inflammation because administration of cerivastatin 2 and 13 h after LPS injection did not protect mice from LPS-induced death (data not shown).

Excessive production of NO by iNOS, together with overproduction of cytokines, is thought to contribute to the pathogenesis of various diseases, including infections, cancers, and autoimmune diseases (Kröncke et al., 1998; Takamura et al., 1998). Therefore, cerivastatin might be useful for the prevention of some of these diseases. Further experimental and clinical studies will be needed to clarify such protective effects of cerivastatin, in fact, of HMG CoA reductase inhibitors.

In conclusion, cerivastatin, an HMG CoA reductase inhibitor, inhibited overproduction of cytokines and NO and improved survival of mice with LPS-induced sepsis. These findings suggest that cerivastatin may be useful for preventing sepsis.