Exacerbation of lymphocytic choriomeningitis in mice treated with the inducible nitric oxide synthase inhibitor aminoguanidine

J of Neuroimmunology Journal of Neuroimmunology 71 (1996) 31–36

Exacerbation of lymphocytic choriomeningitis in mice treated with the . . .. - . . .. mduclble mtrlc oxide synthase inhibitor aminoguanidine Iain L. Campbell * Departntent of Neuropharmacology, The Scripps Research Institute, 10666 North Tm-reyPines Rd. Lu Jolla, CA 92037, USA Received 29 March 1996; revised 18 June 1996; accepted 19 June1996

Abstract To elucidate the possible involvementof the induciblenitric oxide synthase(iNOS) and NO in the developmentof lymphocytic choriomeningitis(LCM), the consequencesof inhibitionof’iNOS by the inhibitor aminoguanidinewas examinedin mice following intracerebralinfection with LCM virus (LCMV). Aminogmmidineadministrationto mice infected with LCMV completelyblocked increased plasma nitrate/nitrite levels and led to increased proinflammatorycytokine gene expression at early stages of lesion developmentin the brain, enhancedclinical severityand decreasedsurvivaltime. The levels of LCMV recoveredfrom the brain of aminoguanidinetreated mice did not differ from those in infectedcontrol mice. These findings argue against either an anti-viralor pathogenicrole of NO in LCM but rather suggesta possibleprotectiveactionof this mediator. Keywords: Lymphocytic choriomeningitis virus; Nitric oxide; Aminoguanidine

1. Introduction Nitric oxide (NO) synthesized by the inducible enzyme, nitric oxide synthase (iNOS), is thought to be a key mediator in the host immune response to infection where it may have protective actions as well as mediate cellular injury (reviewed in Refs. Choi (1993); Lipton and Rosenberg (1994); Moncada and Higgs (1993); Nathan and Xie (1994)). Expression of iNOS can be induced in a wide variety of cells including macrophages (Moncada and Higgs, 1993; Nathan, 1992) and CD4+T~l-lymphocytes (Taylor-Robinson et al., 1994) in the periphery and microglia and astocytes in the central nervous system (CNS) (Boje and Arora, 1992; Galea et aL, 1992; Simmons and Murphy, 1992; Simmons and Murphy, 1993). A variety of mediators including lipopolysaccharide and cytokines such as IFN--y and IL-1 are important signals for the activation of iNOS expression (Moncada and Higgs, 1993; Nathan, 1992). Amongst its many properties NO produced by iNOS has been ascribed a key role in the host anti-viral response to both DNA and RNA viruses (Croen, 1993; Karupian et al., 1993). In fact, induction of NO production from macrophages appears to be one important pathway

* Corresponding author. Tel.: + 1-619-7847092;fax: + 1-619-7847377; e-mail: [email protected]

through which IFN-y mediates its anti-viral effects in vitro and in vivo (Karupian et’al., 1993). Consistent with its role in the host response to infection, cerebral iNOS expression was recently shown to be activated in mice with lymphocytic choriomeningitis (LCM) induced by intra-cranial (ic) inoculation with LCM virus (LCMV) (Campbell et al., 1994b). The response in mice to cerebral infection with LCMV is characterized at the pathologic level by significant recruitment and extravasation of immuno-inflammatory cells to sites of viral replication in the brain — principally involving the meninges, choroid plexus and ependymal membranes (Buchmeier et al., 1980; Doherty et al., 1990). The clinical consequences of this response are profound, with infected mice developing severe tremor, progressing to catastrophic seizure and eventual death around day 6 to day 8 post-infection. Passive-transfer (Baenziger et al., 1986; Dixon et al., 1987) and d-ckpktiort (Leist et al., 1987; Moskophidis et al., 1987) experiments have shown that CD8+-MHC class 1 cytotoxic T-lymphocytes (CTL) but not CD4+ T-lymphocytes are pivotal in the development of LCM. Expression of iNOS in LCM is closely associated with and dependant on the cerebral immunoinflammatory response and is restricted to cells within the inflammatory infiltrates and in proximity to areas of LCMV infection in the brain (Campbell et al., 1994b). Interestingly, the majority of the

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I.L. Campbell/Journal of Neuroimmunology 71 (1996) 31-36

iNOS containing cells in the brain in LCM belong to the Mac-1 positive population and most likely represent infiltrating macrophages while T-lymphocytes belonging to the CDS+ and CD4+ subsets are negative, The kinetics and distribution of cerebral iNOS expression in LCM suggested a major, but undefined role for the iNOS/NO pathway in the pathogenesis of this immune-mediated necrologic disease (Campbell et al., 1994b). In order to elucidate further the involvement of the iNOS/NO response in the development of LCM, in the present study the consequences of inhibition of iNOS by the inhibitor aminoguanidine were examined in mice following ic infection with LCMV.

were killed by anesthetic overdose at day 3 or day 6 post-infection. The brain was immediately removed and bisected sagittally down the mid-line and the left hemibrain immediately frozen in liquid nitrogen and stored at –75°C pending isolation of poly (A+) RNA as described previously (Badley et al., 1988; Campbell et al., 1994a). Aliquots (5 Kg) of poly (A+) RNA sample were subsequently analyzed for cytokine gene expression by RNase protection assay as described previously (Campbell et al., 1994a). The remaining hemibrain was fixed in ice-cold 4% paraformaldehyde in PBS, pH 7.4, embedded in paraffin and 5 pm sagittal sections were cut onto polylysine coated slides and stained with hematoxylin and eosin for routine histological examination.

2. Methods

2.3. Determination of plasma nitrate/nitrite

2.1. Induction

of LCM

and

aminoguanidine administration

Adult (8–10 week old) male Balb/c mice were maintained under pathogen-free conditions in the closed breeding colony of the Scripps Research Institute. LCMV ARM 53b stock was obtained from a triple plaque-purified clone subsequently passaged twice in BHK cells (Dutko and Oldstone, 1983). For induction of LCM, 30 mice were inoculated ic with 25 @ of PBS containing 200 pfu of LCMV. At this dose of LCMV all mice died at day 7. As controls, 12 mice were injected ic with 25 @ of sterile PBS. For administration of aminoguanidine, half of each group of the LCMV-infected and non-infected mice were injected intraperitoneally with 0.5 ml PBS containing 6 mg of aminoguanidine (aminoguanidine hemisulphate; Sigma, St Louis, MO). Mice were administered aminoguanidine every 12 h for up to 7 days commencing at the same time as inoculation with LCMV. The remaining infected and control animals were treated similarly with PBS excluding arninoguanidine. At days 3 and 6 post-infection, 3 mice from each group were anesthetized and blood collected from the brachial artery into heparinized 1.5 ml microfuge tubes. After centrifugation for 10 min at 10,000 rpm, plasma was transferred to clean tubes and stored at –70”C pending assay of nitrate/nitrite (see below). Following blood collection, the brain was removed and immediately frozen in liquid nitrogen and stored at –70”C pending measurement of LCMV titers. A group of 12 mice was also injected ip with 20 ~g LPS in 0.5 ml of PBS. In six of these animals, immediately prior to the injection of LPS, aminoguanidine (6 mg in 0.5 ml PBS) was also administered intraperitoneally. Six hours following LPS injection, all mice were anesthetized, bled and plasma prepared as described above. 2.2. Molecular and cellular pathologic analysis Control mice treated with and LCMV-infected mice treated with or without aminoguanidine as described above

leuels

Immediately prior to assay for nitrate/nitrite, plasma was treated by centrifugal ultrafiltration (Ultrafree-MC Filter Unit. Millipore, Bedford, MA). For nitrate/nitrite measurement, 80 @ of filtered plasma was used for assay employing a commercially available kit (Alexis, San Diego, CA). This assay was performed in accordance with the Manufacturer’s instructions. 2.4. Assay of LCMV in the brain Frozen brain was homogenized in PBS and immediately centrifuged to remove debris. Infectious LCMV present in the clarified homogenate was quantitated by plaque assay on Vero cell monolayer as described (Ahmed et al., 1984).

3. Results and discussion In mice infected ic with LCMV, plasma nitrate/nitrite levels were found to be significantly elevated at day 3 and 6 post-infection (Fig. 1). Initially, following LCMV infection, iNOS gene expression is observed predominantly in the spleen and peaks around day 3, while from day 4 to 6 increasing levels of this enzyme are found in the brain (Campbell et al., 1994b). Therefore, the systemic elevation in nitrate/nitrite levels in LCMV-infected animals observed in the present study, is compatible with the production of NO that follows activation of iNOS gene expression. Aminoguanidine is reportedly a potent and somewhat specific inhibitor of iNOS (Misko et al., 1993) and has been used widely to study the role of the NO generating enzyme in various experimental inflammatory and autoimmune disease models (Corbett et al., 1993; Cross et al., 1994; Iadecola, 1993; Zielasek et al., 1995). The therapeutic regime employed in the present study for the treatment of LCMV-infected mice was comparable to that previously shown to be effective in an EAE model (Cross et al., 1994). The efficacy of the aminoguanidine treatments to

I.L. Campbell/Journal of Neuroimmunology 71 (1996) 31-36 44

* r

I

I

Fig. 1. Effect of aminoguarridineadministration on plasma nitrate\ nitrite levels of mice infected ic with LCMV or injected ip with LPS. Aminoguarridine(6 mg ip per mouse) was given to infected animals at 12 h intervals over the course of the experiment. In the case of LPS, mice received a single dose of aminoguanidine at the time of LPS injection and plasma collected 6 h later. Each column represents the mean+SEM with n = 3 for all the LCMV and normal groups and n = 6 for both LPS groups. For LCMV at each timepoint and LPS plasma nitrate/nitrite levels were significantly different (p <0.05, Student’s t-test) versus normat controls ( *) or aminoguanidine treated (#) animals.

inhibit iNOS activity in mice infected with LCMV or treated with LPS was assessed by determining plasma nitrate/nitrite levels (Fig. 1). In the case of LCMV infection, arninoguanidine administration completely blocked the elevation in systemic nitrate/nitrite. Treatment of mice with LPS resulted in circulating levels of nitrate/nitrite significantly higher than those following LCMV-infection. However, similar to LCMV infection, aminoguanidine administration completely blocked this LPS-induced increase in plasma nitrate/nitrite. These experiments support further the notion that the increased plasma nitrate/nitrite levels following LCMV infection are due to NO production from NOS. Importantly, they also illustrate that the aminoguanidine dosing schedule employed was very effective in inhibiting the activity of iNOS that accompanied LCMV infection. The temporal and spatial expression of iNOS activation in the brain following ic LCMV infection highlights the possible involvement of NO in the pathogenesis of LCM where, amongst other things, it has been suggested NO may be neurotoxic and contribute to the necrologic collapse in this disorder (Campbell et al., 1994b). Aminoguanidine blockade of iNOS was therefore utilized as an approach to dissect out the role of the iNOS/NO response in the development of LCM. Mice infected ic with LCMV were treated aggressively with arninoguanidine and their clinical status carefully monitored and compared with similarly infected animals given buffer alone (Fig. 2). LCMV-infected animals given buffer alone exhibited signs of LCM (shivering and ruffled fur) by day 6 post-infection and all died 24 h later with convulsions and

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seizure. Administration of arninoguanidine to LCMV-infected mice resulted in an earlier onset of more severe illness by day 5 post-infection with 50% of animals dying by day 6 and the remainder dying by day 7 post-infection. Therefore, iNOS blockade with aminoguanidine results in exacerbation of clinical LCM. These findings do not support a neurotoxic role for NO in LCM — on the contrary, they indicate NO may have protective actions. This latter interpretation however is complicated by the observation that while administration of aminoguanidine in non-infected control mice was not lethal, the presence of ruffled fur and diarrhea was noted in this group compared with non-infected mice given buffer alone (Fig. 2) suggesting a possible toxic side-effect of the drug treatment. Side-effects of aminoguanidine therapy have not been noted in other experimental studies in mice (Corbett et al., 1993; Cross et al., 1994). However, the possible existence of such side-effects as indicated by the present study, means that negative interactions of the drug not related to its iNOS inhibiting function cannot be ruled out in contributing to exacerbation of LCM. We determined if the clinical exacerbation of LCM following aminoguanidine treatment correlated with increased severity of pathological alterations in the brain. Routine histological examination of brain failed to reveal a significant difference in the extent of mononuclear cell infiltration between treated and non-treated LCMV-infected animals (data not shown). In parallel studies, tie functional status of the immunoinflammatory response in these mice was examined by determining the level of expression of a number of key proinflarnmatory cytokine genes that had been shown previously to be upregulated in the brain in LCM (Campbell et al., 1994a). Confirming this previous report, in the present study, elevated expression of TNF-a, IL-a and IL-1/3 mRNA was discernible by day 3 post-infection and further increased by day 6 (Fig. 3A and B). While at day 6 post-infection, high levels of IFN-y mRNA were also observed. Treatment of

% Survival/CIlnioalseora Day -

1234567

unlnfectd

El-

- ‘ --

unhrfected+AG

El+

+ ‘+’

LCMV

El+

+ ‘+;”

LCMV+AG

m b

-

Fig. 2. Survival times and clinical scores in LCMV infected mice treated with or without aminoguaoidine. For clinicat scores: – = no illness, + = ruffled fur, + + = shivering and ruffled fur, + + + = convulsions and seizures.

I.L. Campbell/Journal of Neuroimmunology 71 (1996) 31-36

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LCMV-infected mice with aminoguanidine resulted in significantly higher levels of expression of the TNF-a, IL-1a and IL-1 @ mRNAs at day 3 post-infection. However, at day 6 post-infection, there was no significant difference between the treated and non-treated LCMV-infected mice in the level of expression of these cytokine transcripts nor those for IFN--y. These findings indicate that while iNOS inhibition in LCM did not influence the degree of recruitment of immuno-inflammatory cells to the brain, it was associated with increased functional activation of early infiltrating cells. This latter observation is consistent with recent evidence that implicates NO as an important autocrine negative feedback regulator during microphage activation and function (Florquin et al., 1994; Sicher et al., 1994). Such a mechanism for microphage control however did not significantly influence the late stage of LCM where cytokine gene expression was not altered by arrtinoguanidine administration. The explanation for this is not known. One possibility however, is that the high levels of IFN--y present at this time may totally override the counterregulatory actions of NO on the microphage and/or other inflammatory cells. While we did not determine the identity of the cells responsible for the increased expression of the proinflammatory cytokines early in LCM, the nature of these cytokines is suggestive of a microphage source. The increased functional activation of these cells at this time in the development of LCM might indicate and result in earlier onset of tissue injury in the aminoguanidine treated animals and thereby contribute to the clinical exacerbation of this disease. A prominent function of NO that may contribute to the host protective state in infection is its ability to inhibit the replication of certain classes of viruses (Croen, 1993;

A. TNF-~ TNF-o IL-la IFN-y

IL-1P

Fig. 4. LCMV titers in the brain of mice treated with or without aminoguanidine. Mice were killed at the times shown and the brain removed and immediately frozen and stored at –70°C pending measurement of infectious virus titer by plaque assay on Vero cells. Each column represent the mean+ SEM (n= 3).

Karupian et al., 1993). In considering this point, it is feasible the exacerbation of LCM produced by aminoguanidine treatment may be due to removal of antiviral NO from the host response with consequent amplification of LCMV replication. This possibility was addressed by analyzing the levels of LCMV present in the brain of aminoguanidine or buffer treated mice (Fig. 4). The findings indicated that the levels of LCMV in the brain did not differ significantly between the aminoguanidine and buffer treated groups at day 3 or day 6 following infection. Thus, aminoguanidine treatment does not lead to significant changes in the level of LCMV in the primary tissue compartment in which this virus replicates (i.e. the

B. Day 3 I 6 I 180I 160I s I ~ 140;120 -

;

2100+ Z 80-

I

u ~ 60E 40-

I I

316 I I I

I I I I I

363

I

x 1

I I

20 L32

0 TNF-~ “ lltF-a

❑ Control+AG

“ IL-1a “ IL-1 Cytokine

❑

LCMV

IFN-y

■ LCMV+AG

Fig. 3. Cytokine gene expression in the brain. Mice (control or LCMV-infected) were given aminoguanidine or saline vehicle as described in Section 2. Animals were killed on day 3 and 6, the brain removed, immediately frozen and poly (A+) RNA prepared. The level of cytokine gene expression in each sample was determined by RNase protection assay using 5 pg of RNA. A representative autoradiogram from one experiment is shown (A). Quantitative analysis (B) was done by densitometry using NIH Image 4.7. The values shown represent the mean + SEM for three independent samples analyzed in each group. In the case of the control samples only the mean is shown since one of the samples was below the detectable range for densitometry. Significance for LCMV + AG versus LCMV. (*) p <0.05 (Student’s t-test)

I.L, Carrrpbell/Jrrurnalof Neuroimmwsology 71 (1996) 31-36

brain), following ic inoculation. This finding suggest that LCMV replication is not sensitive to inhibition by NO and moreover, exacerbation of LCM by aminoguanidine is not consequent to increased levels of LCMV in the brain. Interestingly, exacerbation of myelin basic protein (MBP) induced-EAE was recently reported in Lewis rats given aminoguanidine (Zielasek et al., 1995). Although the pathogenesis of this disorder is quite different to LCM, the similar outcomes resulting from aminoguanidine treatment in the two models suggests a possible common mechanism of action (e.g. altered microphage regulation as discussed above) of the NOS inhibiting agent. However, in difference to this idea, administration of aminoguanidine was reported to ameliorate rather than exacerbate MBP-EAE in SJL mice (Cross et al., 1994). Since the doses of amirmguanidine and treatment protocols used in the varous studies were quite similar the dichotomy in the results is puzzling, While this may reflect other experimental and technical differences it also highlights the possible complexity of NO actions in different neuroinflammatory lesions. The recent development of mutant mice with tatgeted disruption of the microphage iNOS gene (MacMicking et al., 1995; Wej et al., 1995) will help to resolve some of these issues and remove the need for the use of agents such as aminoguanidine. In conclusion, administration of the semi-specific iNOS inhibitor aminoguanidine to mice infected with LCMV exacerbated LCM without altering virus levels in the brain. These findings do not support an anti-viral or pathogenic role of NO in LCM, rather they suggest a possible protective action of this mediator. Acknowledgements The author thanks Anna Samimi for technical assistance and Monte Hobbs for providing the ML-1 1 probe set used in the RNase protection assays. These studies were supported by U.S. Public Health Service Grant MH 50426. This is manuscript number 9993-NP from the Scripps Research Institute. References Ahmed, R., Salmi, A., Butler, L.D., Chiller, J.M. and Oldstone, M.B.A. (1984) Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. J. Exp. Med. 60, 521-540. Badley, J.E., Bishop, GA., St. John, T. and Frelinger, J.A. (1988) A simple, rapid method for the purification of poly A+ RNA. Biotechniques 6, 114–1 16. Baenziger, J,, Hengartner, H., Zinkernagel, R.M. and Cole, G.A. (1986) Induction or prevention of immunopathologic disease by cytotoxic T cell lines specific for lymphocytic choriomeningitis virus. Eur. J. Immunol. 16, 387–393. Boje, K.M. and Arora, P.K. (1992) Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res. 587, 250-256.

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