Dose-dependent attenuation of lipopolysaccharide-fever by inhibitors of inducible nitric oxide-synthase in guinea pigs

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Abstract

Different doses of aminoguanidine or S-methylisothiourea, both predominantly inhibitors of the inducible form of nitric oxide (NO)-synthase, were injected into the arterial circulation of guinea pigs alone or along with 10 μg/kg bacterial lipopolysaccharide. Doses of 10 mg/kg, 50 mg/kg or 250 mg/kg aminoguanidine per se had no influence on abdominal temperature of guinea pigs. Only the highest dose of aminoguanidine (250 mg/kg) completely suppressed the first phase of the biphasic febrile response to lipopolysaccharide-injections. Lipopolysaccharide-fever was not modulated by administration of 10 mg/kg or 50 mg/kg aminoguanidine, when compared to fever in response to injections of lipopolysaccharide along with solvent. Doses of 10 mg/kg or 50 mg/kg S-methylisothiourea did not alter abdominal temperature while a dose of 250 mg/kg S-methylisothiourea had a lethal effect in guinea pigs. The febrile response to lipopolysaccharide was unimpaired by administration of 10 mg/kg S-methylisothiourea, while a dose of 50 mg/kg again attenuated fever predominantly by a suppression of the first fever phase. None of the applied doses of aminoguanidine or S-methylisothiourea resulted in a significant attenuation of the lipopolysaccharide-induced circulating cytokines tumor necrosis factor-α (TNF-α) and interleukin-6. The drugs themselves, without lipopolysaccharide-injections, did not enhance or reduce circulating levels of the investigated cytokines. The results indicate that endogenous NO may participate in the induction of lipopolysaccharide-fever and that fever suppression by systemic administration of NO-synthase inhibitors occurs independently from the lipopolysaccharide-induced circulating cytokines.

Introduction

Microbial products such as lipopolysaccharide from gram-negative bacteria exert their biological effects in a host organism by the induction of a cytokine cascade (Dinarello, 1991). The cytokines themselves induce a number of enzymes including the inducible form of nitric oxide (NO)-synthase (iNOS). Therefore, the question arises if well-known physiological responses which accompany the activation of the cytokine cascade are, in part, mediated by the cytokine-induced enzyme iNOS. One of the most common responses to infection or inflammation is the development of a febrile response and there is agreement that representative members of the cytokine cascade are involved in the generation of fever Kluger, 1991, Kozak et al., 1995, Chai et al., 1996, Rothwell, 1997, although especially the early phase of fever is possibly induced without direct participation of cytokines (Blatteis and Sehic, 1997).

Does NO, an important signal created by cytokines, play a role in the manifestation of the febrile response? Recent studies with the aim to obtain an answer to this question were leading, in part, to conflicting results. Some experiments in rabbits suggest that NO might have antipyretic properties Gourine, 1995, Weihrauch and Riedel, 1997. In cats, NO seems to have no effect on fever at all (Redford et al., 1995). Other studies performed with rats Reimers et al., 1994, Scammell et al., 1996, Roth et al., 1998b, pigs (Parrott et al., 1998) or guinea pigs (Roth et al., 1998c) provide evidence that NO participates in the development of a febrile response. The suggestion for a propyretic role for NO is predominantly based on the effects of systemic administrations of N-nitro-l-arginine-methylester (l-NAME) along with pyrogens, a substance which is regarded as a rather unspecific inhibitor of all isoforms of NO-synthases. Injections of l-NAME directly into the circulation result in attenuation of fever, but this drug per se also induces hypothermia when administered at higher doses Scammell et al., 1996, Roth et al., 1998c. Although the results of the studies in rats, pigs and guinea pigs suggest that NO might play a role in the generation and maintenance of fever in these species of experimental animals, some questions still remain open: (A) Is the hypothermia which is observed after administration of l-NAME at higher doses due to inhibition of NO-synthesis or is it a drug-effect? (B) Do specific inhibitors of iNOS, the only form of this enzyme which is induced by lipopolysaccharide or cytokines, influence fever in a similar way as the unspecific inhibitor l-NAME? (C) Is there a positive or negative feedback of iNOS inhibitors on the lipopolysaccharide-induced cytokine network which could be responsible for possible modulations of the febrile response? Therefore, we injected different doses of aminoguanidine or S-methylisothiourea, both predominantly inhibitors of iNOS Szabo et al., 1994, Zhao et al., 1996, into the arterial circulation of guinea pigs alone or along with lipopolysaccharide and investigated their influence on body temperature and on circulating cytokines.

Section snippets

Animals

This study was performed in male guinea pigs with a body mass in the range of 385–410 g at the time of surgery. The animals were housed in individual cages at 22°C and a 12:12 h light:dark cycle (light off at 0700 h). The animals had access to food and water ad libitum. Twice a week the reservoires were filled with fresh pelleted food and water and at the same time the cages were changed. At least one week before the experiment was performed, the animals were prepared surgically (see below).

Results

The effects of intra-arterial injections of 10 μg/kg lipopolysaccharide along with solvent or different doses of aminoguanidine (10 mg/kg, 50 mg/kg or 250 mg/kg), or of solvent and the three doses of aminoguanidine alone on abdominal temperature of guinea pigs are summarized in Fig. 1a and b.

Lipopolysaccharide-induced fever was not modulated by administration of 10 mg/kg or 50 mg/kg aminoguanidine. In the group of guinea pigs which received 250 mg/kg aminoguanidine along with

Discussion

The main finding of this study is that the iNOS inhibitors aminoguanidine and S-methylisothiourea cause a dose dependent suppression of the first phase of the biphasic lipopolysaccharide-fever in guinea pigs without any influence on lipopolysaccharide-induced amounts of circulating cytokines, putative mediators of the febrile response. Further, in contrast to the unspecific NOS inhibitor l-NAME, both drugs per se did not cause hypothermia. A number of questions may arise concerning this, in

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft (DFG). We thank Dr. Stephen Hopkins (University of Manchester, UK) for providing us with the WEHI 164 subclone 13 and the B9 cell lines and informing us about important details of the cytokine assays.

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