Department of Microbiology, University of Western Australia, Nedlands, WA 6907, Australia1
Author for correspondence: Nadezda Urosevic. Fax +61 8 9346 2912. e-mail nadia{at}cyllene.uwa.edu.au
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Three different types of nitric oxide synthase have been identified: neuronal (NOS1), macrophage (NOS2) and endothelial (NOS3) (Nathan, 1992 ). Of these, the genes encoding NOS1 and NOS3 map to distal and proximal regions of mouse chromosome 5, respectively (Gregg et al., 1995
; Lee et al., 1995
), and are constitutively expressed in the mouse brain and in endothelial cells, respectively (Bredt et al., 1991
; Lowenstein et al., 1992
). While both NOS1 and NOS3 enzymes are Ca2+ and calmodulin regulated, the third enzyme, NOS2, which is located on chromosome 11 and expressed in macrophages, is inducible and Ca2+/calmodulin independent (Bredt et al., 1991
; Xie et al., 1992
; Gerling et al., 1994
). All three enzymes, although differently regulated, catalyse the conversion of arginine to citrulline with NO as a major product. NO is an active molecule and a potent effector implicated in a variety of biological functions ranging from peripheral vasodilatation and platelet aggregation to neuromodulation and neurotransmission in the CNS (Nathan, 1992
). Furthermore, there is recent evidence suggesting that NO exerts antiviral activity both in vitro and in vivo, possibly contributing to innate immune responses to various viruses including flaviviruses (Karupiah et al., 1993
; Lin et al., 1997
; Reiss & Komatsu, 1998
). NO exhibits a number of properties which could be advantageous to its innate antiviral activity. Among these are its ability to penetrate cells and spread easily between neighbouring cells, the independence of its action from the acquired immune responses and the inability of viruses to develop resistance to such a small compound (Nathan, 1992
; Karupiah et al., 1993
).
To determine if NO is involved in flavivirus resistance controlled by Flv, we have monitored the brain tissue levels of NO before and after flavivirus infection in flavivirus-susceptible C3H/HeJARC (Flvs) mice and in two congenic, flavivirus-resistant mouse strains, C3H.PRI-Flvr and C3H.M.domesticus-Flvr-like. Inbred C3H/HeJARC mice are a lipopolysaccharide (LPS)-responsive subline of the LPS-unresponsive C3H/HeJ mouse strain, which has been maintained as a separate colony in Western Australia for more than 28 years (Silvia & Urosevic, 1999 ). These mice were used as an acceptor strain for the flavivirus resistance Flvr-like allele of wild Mus domesticus during the creation of the flavivirus-resistant mouse strain C3H.M.domesticus-Flvr-like (Urosevic et al., 1999
). The other resistant mouse strain, C3H.PRI-Flvr, was developed by backcross breeding of resistant PRI mice to mice of C3H/He background more than 30 years ago (Groschel & Koprowski, 1965
). These two resistant mouse strains are congenic to C3H/HeJARC mice, and they carry chromosomal segments encompassing Flv which are of different genetic origins (Urosevic et al., 1999
). In addition to Flv, these polymorphic chromosomal segments also encompass Nos1, while Nos2 and Nos3 are located within the non-polymorphic chromosomal segments which are of the same genetic origin (The Mouse Genome Database, The Jackson Laboratory, Maine, USA. Website: www.informatics.jax.org; visited March 2000; Flaherty, 1981
). While there is evidence suggesting the existence of different Flv alleles in these three congenic mouse strains (Sangster et al., 1993
, 1998
; Urosevic et al., 1999
), it is not known whether similar allelism at the Nos1 locus may exist, conferring various levels of basal NO synthesis in the brains of these mice. Furthermore, it is not known whether Flv itself exerts any influence upon NO synthesis controlled by the inducible Nos2 gene.
In order to answer these questions we initially monitored the levels of NO synthesis in the brains of these three congenic mouse strains before and after flavivirus infection. Mice of the C3H/HeJARC, C3H.PRI-Flvr and C3H.M.domesticus-Flvr-like strains were injected intracerebrally (i.c.) with either a mouse osmolality phosphate-buffered saline (MOBS) or 105·2 infectious units (IU) of Murray Valley encephalitis (MVE) virus strain OR2. At various days post-infection (p.i.), groups of three to five mice of each strain were sacrificed and used to monitor both virus titres and NO levels in brain tissue homogenates (1:10, w/v). Virus titres were determined by 50% tissue culture infective dose (TCID50) assay and were shown to be significantly lower in the brains of flavivirus-resistant than in the brains of flavivirus-susceptible mouse strains (Fig. 1A). In contrast, the brain tissue NO levels, as determined by indirect measurement of the stable nonvolatile breakdown product nitrite (
) using the Griess reagent (Promega) did not show any significant difference between susceptible and resistant mouse strains before and after virus infection (Fig. 1B
). This indicates that different levels of flavivirus replication observed in the brains of susceptible and resistant mice, which are controlled by Flv, do not correlate with the extent of either constitutive or inducible NO synthesis in the brains of these mice.
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The tight regulation of NO synthesis in the brain is not surprising considering the importance of its role in neurotransmission and neuromodulation within the CNS. In contrast, NO synthesis in macrophages is induced by various stimuli, including bacterial LPS, interferon- and different combinations of these agents with tumour necrosis factor (TNF)-
and -
, interferon-
and -
, and IL-1 (Nathan, 1992
). It has been suggested that NO is an effector molecule mediating cytotoxic actions of macrophages against tumour cells and micro-organisms (Xie et al., 1992
). Since its production in macrophages is controlled by the high output pathway (MacMicking et al., 1997
), NO may represent one of the major mechanisms by which virus replication in macrophages is controlled.
Peritoneal macrophages from flavivirus-resistant mice have been shown to be a major in vitro model of the Flv-controlled virus resistance in addition to mouse embryo fibroblasts (Goodman & Koprowski, 1962 ; Brinton, 1983
; Silvia et al., 1997
). In the current study we have monitored the in vitro effect of virus replication on NO production in mouse macrophage cultures derived from flavivirus-resistant and -susceptible mice to assess whether Nos2 is implicated in innate Flv-controlled resistance observed in vitro (Silvia et al., 1997
). Peritoneal macrophages lavaged 3 days after intraperitoneal injection of mice with thioglycollate were treated with E. coli K235 LPS (Sigma) for 16 h before being infected with West Nile (WN) encephalitis virus strain Sarafend at an m.o.i. of 1. Three to five aliquots of replicate tissue culture supernatants were initially removed at 24 h post-treatment (p.t.) and then at every day afterwards for up to 8 days for determination of virus titres by TCID50 (Fig. 3
) or
levels with Griess reagent (Fig. 2
), respectively. While LPS produced a stimulatory effect on NO production in macrophages derived from all three congenic C3H mouse strains at every day p.t. for up to 8 days starting at day 1, infection with WN virus alone did not significantly affect basal NO levels at any of these time-points (Fig. 2
).
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When the LPS pre-treated macrophages derived from these three congenic LPS-responsive mouse strains were infected with the flavivirus WN virus strain Sarafend for 1 h, further changes in NO production were observed (Fig. 2). While in the absence of LPS treatment WN virus alone did not significantly affect NO production in macrophages of all three mouse strains, it caused significant changes in NO production of LPS pre-treated cells (Fig. 2
). Infection with WN virus caused either a significant increase (Students t-test, 0·002>P>0·001) in NO production in cells derived from C3H/PRI-Flvr mice or a decrease in NO production in the cells derived from C3H/HeJARC and C3H.M.domesticus-Flvr-like mice (0·002>P>0·001 and 0·10>P>0·05, respectively) (Fig. 2
). A similar modifying effect of viral infection on NO production in mouse macrophages has been previously reported for another flavivirus, tick-borne encephalitis virus, and this effect was shown to be mediated by
/
-interferons (Kreil & Eibl, 1995
). While it is not likely that
/
-interferon genes are differently regulated between C3H congenic mouse strains, as illustrated by the similar brain interferon levels (Hanson et al., 1969
), it is more likely that the adverse effect of viral infection on LPS-stimulated NO production is mediated by some chromosome 5 locus other than Flv. Since both the LPS signalling pathway (Han et al., 1994
) and a cascade of events leading to induction of iNOS in macrophages (Gao et al., 1998
) are very complex, it is difficult to predict from the current study which step in this pathway is responsible for the difference observed. Consequently, our congenic C3H mouse strains may provide an excellent model to further dissect effects of LPS and virus on NO production in vitro.
In the current study we have also observed that the in vitro pre-treatment with LPS of macrophages from both flavivirus-resistant and -susceptible mice has restricted the replication of WN virus in these cells (Fig. 3). While there was still some residual virus replication observed in the cells derived from susceptible (C3H/HeJARC) mice, LPS stimulation completely obstructed WN virus replication in primary macrophages derived from flavivirus-resistant C3H/PRI-Flvr and C3H.M.domesticus-Flvr-like mouse strains (Fig. 3
). This outcome may have resulted from either the decreased susceptibility of activated macrophages to virus infection, or may have been mediated by an antiviral compound such as NO. The inhibitory effect of NO on flavivirus RNA replication, protein synthesis and release from the monocyte/macrophage cell lines has been previously reported for Japanese encephalitis virus (Lin et al., 1997
). However, regardless of what mechanism is responsible for this antiviral effect, it does not appear to abrogate Flv regulated effects on flavivirus replication.
In conclusion, data presented here indicate that NO is not implicated in Flv-controlled resistance in mice. This has been supported by the lack of NO responses to flavivirus infection in both mouse brain tissue and peritoneal macrophages in flavivirus-resistant and -susceptible mice before and during the acute phase of infection. While the virus titres in the brains of susceptible mice were significantly higher, resulting in much greater neuronal damage and more severe tissue inflammation than in the brains of resistant mice (data not shown), no significant effect of the virus on the brain tissue NO levels was observed in these mice. We have also presented some additional evidence suggesting the involvement of chromosome 5 genetic loci other than Flv in the control of LPS-induced NO responses in mouse peritoneal macrophages. This evidence suggests that natural resistance to flaviviruses and LPS-induced antiviral state are two distinct mechanisms, which involve independent signalling pathways in the cell.
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References |
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Received 17 May 2000;
accepted 16 November 2000.