Melatonin prolongs survival of immunodepressed mice infected with the Venezuelan equine encephalomyelitis virus

Ernest0 BonillalJ , Carolina Rod6n’ , Nereida Valero’ , HBctor Pons3, Leonor Chacin-Bonilla’ , Jorge Garcia Tamayo4, Zulay Rodriguez *, Shirley Medina-Leendertz2 and Florencio Aiiez’ ‘Znstituto de Znvestigaciones Clinicas, Universidad de1 Zulia, Maracaibo, Venezuela; ‘ZNBZOMED, FUNDACZTE-ZULZA, Maracaibo, Venezuela; 3Centro de Cin&a Experimental, Universidad de1 Zulia, Maracaibo, Venezuela; 41nstituto Anatomopatoldgico, Universidad Central de Venezuela, Caracas, Venezuela

Abstract Male albino mice immunodepressed after the injection of dexamethasone (DEX) were inoculated intraperitoneally with the Guajira strain of Venezuelan equine encephalomyelitis (VEE) virus. Melatonin (MLT) was administered daily, at a dose of 500 rig/kg bodyweight, for 3 days before virus inoculation and 10 days after. Serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-Z) were determined in all the experimental groups (control, DEX, DEX+MLT, DEX+VEE, DEX+VEE+MLT, VEE and MLT). At day 6 after the virus inoculation, the survival rate was significantly increased from 0% in group DEX+VEE to 32.5% in the group of immunodepressed infected mice treated with MLT (DEX+VEE+M.LT). By day 10 a survival rate of 10% was found in group DEX+VEE+MLT and 0% in group VEE. No alterations in IL-2 serum levels were observed. MLT increased GM-CSF in control and in DEX-treated mice. In the VEE virus-infected mice treated with DEX, serum levels of GM-CSF increased progressively from day 1 to 5 postinoculation. In contrast, the levels of GM-CSF in infected immunodepressed mice treated with MLT decreased significantly from day 1 to 5 postinoculation. At day 5 after viral inoculation, no differences were detected in the cerebral viral titres in groups VEE, DEX+VEE and DEX+MLT+VEE. These results show that MLT does not inhibit VEE viral replication in the brain of immunodepressed mice.

Keywords:Venezuelan equine encephalomyelitis, experimental model, mice, immunosuppression, dexamethasone, melatonin, GM-CSF, interleukin-2

Introduction The pineal gland is a neurochemical transducer cap-

able of perceiving basic environmental information and integrating it with the synthesis and release of a variety of hormones of which melatonin (MLT) or N-acetvl- 5methoxytryptamine is the most‘ thoroughly studied (AXELROD, 1974). MLT appears to be involved in synchronizing the circadian and seasonal timing of several physiological and behavioural processes (BINK- LEY, 1 ~~~;LIEBERMAN etal., 1984; REITER~Z~~., 1984) and it seems to be a powerful hydroxyl-radical scavenger which provides on-site protection against oxidative da- mage to cell components (REITER et a1.,1995). The immunoenhancing effects of MLT are becoming in- creasingly clear. By binding to T-helper cells, MLT gives rise to a series of events leading to an increase in immune response (MSTRONI, 1995): Inhibition of MLT synth- esis and abrogation of the cvclicitv of MLT secretion bv evening admimstration of P-blockers lead to a significant depression of humoral and cell-mediated immune re- sponses in rodents (RADOSEVIC-STASIC et al., 1983; MAESTRONI et al., 1986). When chronically injected into young mice or mice immunodepressed by ageing or by cyclophosphamide, MLT was able to enhance the anti- body response to a T-dependent antigen (CAROLEO et al., 1994).

Viral infection can produce an increase in circulating glucocorticoids (BESEDOVSKY~ZDELREY, 1989;B~~- HUR et al., 1995). On the other hand, treatment with corticosterone of mice infected with the non-invasive, attenuated encephalitic virus West Nile resulted in high viraemia and a marked increase in mortality when com- pared to control untreated mice (BEN-NATHAN et al., 1996). A protective effect of MLT in dexamethasone (DEXI-treated mice infected with the West Nile virus &asreported(B~~-N~~~~~ etal., 1995).MLThasalso been shown to protect mice infected with the highly lethal Venezuelan equine encephalomyelitis (VEE) virus

Author for correspondence: Dr Emesto Bonilla, Instituto de Investigaciones Clinicas, Facultad de Medicina, Universidad de1 Zulia, Apartado Portal 115 1, Maracaibo, Venezuela; phone +58 61 597247, fax +58 61 835587, e-mail ebonilla@iamnet.com

by postponing the onset of the disease and death by several days (BONILLA et al., 1997).

In order to evaluate the participation of the immune system in the protection of VEE virus-infected mice by MLT, we determined the survival rate in immunode- pressed mice infected with the VEE virus and treated with MLT and the effects of this neurohormone on the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 ( IL-2 ).

The present work demonstrates that MLT prolongs survival of immunodepressed mice infected with the VEE virus.

Materials and Methods Male albino mice, NMRI-IVIC strain from the Vene-

zuelan Institute for Scientific Research (IVIC), weighing 25-30 g and fed ad libitum with laboratory chow and tap water, were maintained in a room with controlled tem- perature (24°C) under a 12-h light/dark cycle. The VEE virus stock used for experiments was prepared in Vero cells and contained 6.8 X 10’ plaque-forming units per mL (PFU/mL). Mice were inoculated intraperito- neally with 0.3 mL containing 10 LD50 (100 PFU) of the Guajira strain of VEE virus suspended in 0.4% bovine albumin borate-buffered saline solution (BABS) (HAMMON &SATHER,~~~~).

Melatonin (Research Biochemicals International, MA, USA) at a dose of 500 l.tg/kg bodyweight was diluted in saline and injected daily subcutaneously, at 17:00, starting 3 days before and continuing to 10 days after virus inoculation (BONILLA et al., 1997).

Dexamethasone (Sigma Chemical Co., St Louis, MO, USA), at a dose of 5 mg/kg intramuscularly, was diluted in saline and administered daily starting IO days before virus inoculation and continuing through 4 days after inoculation in order to induce immunodepression.

Virus content in brain was plaque assayed in chicken embryo fibroblasts (BERGOLD-& ti~~~ti, 1968). Serial dilutions of VEE virus were added to confluent mono- layers in 24-well culture plates which were incubated at 37°C for 2 h for viral adsorption. The standard overlay (Eagle’s medium 2 X and 0.5% agarose) was added. Plates were incubated at 37°C in 5% CO, for 48 h (until cytopathology was evident). Plaques were counted after staining monolayers with 0.2% crystal violet.

208 ERNEST0 BONILLA ETAL.

The quantitative determination of mouse IL-2 and Table 1. Serum levels of IL-2 in immunodepressed GM-CSF in sera was based on a solid-phase ELISA mice infected with the Venezuelan equine ence- system developed by Amersham International plc (Bio- trak TM).

phalomyelitis (VEE) virus and treated with mela- tonin

Animals were divided in 7 experimental groups: con- trol, DEX, DEXtMLT, DEXfVEE, DEX+MLT+ VEE, VEE, and MLT. Treatment

Serum levels of IL-2 bg/mL)

Mice were sacrificed in groups of 3 starting the same day of the viral inoculation and then 1,3,5 and up to 10 days post-inoculation. Blood was collected by cardiac puncture and sera were stored at -70°C until analysed. The brains were suspended (20% w/v) in 50 mM bluffer Tris-HCl oH 8.0.

Control 98.6 3~ 14.0 DEX 111.9 i 36.9 DEX + MLT 64.6 f 20.5 DEX + VEE 68.1 i 46.7 DEX+VEE+MLT 91.1 f 13.2 VEE 122.1 * 21.5 MLT 116.0 & 25.6

Values are expressed as mean f SEM. Dexamethasone (DEX; 5 mg/kg bw) was administered daily during 10 days before and 4 days after the infection. Melatonin (h4LT; 500 pg/kg bw) was injected daily starting 3 days before the infection and continuing through 4 days after infection. On the fifth day the IL-2 serum levels were determined.

Data weie expressed as mean f SEM and were studied by means of the analysis of variance and the Bonferroni’s multiple comparison test where appropriate. The sig- nificance between 2 specific means was determined by a 2-tailed Student’s t test. Differences were considered stastistically significant when P cO.05.

Results At day 6 after the virus inoculation, the survival rate

was significantly increased (P <O-05) from 0% in group DEX+VBE to 325% in group DEX+VEE+MLT. A final survival rate of 10% was fo;nd in immunodepressed infected mice treated with MLT and 0% in infected animals. In addition, 78% of the uninfected mice treated with DEX and 100% of those receiving MLT survived throughout the study (Figure).

In order to confirm the immunodepression induced by DEX, the concentration of lymphocytes and granulo- cytes in blood was determined. On the third and seventh day of treatment with DEX a significant increase in granulocytes (P CO.00 1) and a decrease in lymphocytes (P <O.OOl) were detected.

No significant differences were found in serum IL-2 levels among the groups of mice (Table 1).

The administration of MLT increased serum levels of GM-CSF in control and in DEX-treated mice on days 1, 3 and 5 postinoculation (Table 2). However, in infected immunodepressed mice treated with MLT (DEX+VEE+MLT) GM-CSF levels increased on the first day but decreased significantly on days 3 and 5 when compared with their corresponding controls (DEX+VEE) .

Dexamethhsone treatment did not affect GM-CSF levels when compared to controls on days 1, 3, and 5 postinoculation. -

In VEE virus-infected mice, serum levels of GM-CSF were found to be increased od days 3 and 5 postinocula-

125,

2 loo-

; 75-

f 50-

’ 25-

o- I I I , I , , ) , , ,,

0 12 3 4 5 6 7 8 91011 Days after virus inoculation

-O-MT-O-DWL 4DEX+VBE+MLT+k-“EE -.-DEX+“EE

Figure. Survival rate of immunodepressed mice infected with Venezuelan equine encephalomyelitis virus (VEE) and treated with melatonin (h4LT). Male albino mice immunodepressed after the injection of dexamethasone (DEX) were inoculated intraperitoneally with 10 LD,, of the Guajira strain of VEE virus. MLT (500 pg/kg bw) was administered daily, 3 days before virus inoculation and 10 days after. The survival rate was determined daily, in all experimental groups (20 mice per group); *P CO.05 when compared to DEX+VEE.

tion when compared to control non-infected mice (Table 2). In infected animals treated with DEX (VEE+DEX), an increase in serum GM-CSF content was detected on the third and fifth day postinoculation when compared with the infected mice (VEE).

It is interesting to note that in VEE-infected mice treated with DEX the serum levels of GM-CSF increased progressively from day 1 to day 5. In contrast, in the infected immunodepressed mice treated with MLT (DEX+VEE+MLT) the values of GM-CSF decreased significantly from day 1 to day 5.

On the fifth dav after inoculation. no differences were detected in brah virus levels between the infected (8.56 ZIZ 0.14 log,, PFU/g), the infected immunode- pressed (8.73 rt 0.07), and the infected immunode- pressed mice treated with MLT (8.64 & 0.12).

Discussion The data shown in this work demonstrate that MLT

administration prolongs survival of immunodepressed mice infected with the VEE virus since 6 days after virus inoculation the survival rate was increased from 0 to 32.5%. These results confirm the findings obtained in immunocompetent mice infected with VEE virus and treated with &JILT (500 pg/kg). In the latter the survival rate increased from 0% to 60% (BONILLA et al., 1997). Thk protection provided by ML? to infected i&mu&- depressed mice is approximately half of that given to immunocompetent mice suggesting that this neurohor- mone requires, at least partially, the integrity of the immune system to perform its protective effect. It has been shown that MLT also has a protective effect in DEX-treated mice infected with the attenuated non- invasive West Nile virus (BEN-NATHAN et al., 1995). This virus does not invade the brain but it can induce encephalitis in immunodepressed mice. In contrast, the VEE virus is highly lethal and in immunocompetent mice infected with this virus, paralysis, coma and death occur usually by the sixth day after inoculation (BONILLA et al., 1988). The decrease in the survival rate of the immuno- depressed infected mice (DEX+VEE) as compared to the immunocompetent infected mice (VEE) suggests that an active immune system is necessary to retard the death of the animals.

A significant increase in GM-CSF synthesis and/or release oroduced bv MLT on healthv control mice has been p&iously reported (MAESTRONI et al., 1994a). It has been shown that MLT can protect haematopoietic functions in mice from the toxic effect of cancer chemo- therapy compounds without interfering with their anti- cancer action (MAESTRONI et al., 1994b). This effect appeared to involve bone-marrow T-cells and the en- dogenous production of a colony-stimulating factor

MELATONINANDVENEZUELANEQlJINEENCEPHALOMYELITISVIRUS 209

Table 2. Serum levels of granulocyte-macrophage colony-stimulation factor (GM-CSF) in immunodepressed mice infected with the Venezuelan equine encephalomyelitis (VEE) virus and treated with melatonin

Days post-inoculation

Treatment 1 3 5

Control 5.64 zt 0.97 6.70 zt 1.48 5.18 i 1.02 DEX 7.45 It 0.55 787 * 0.85 7.58 f 0.88 DEXfMLT 10.29 zt 0.87b,c 13.88 f 0.95a’bac 13.93 f 0.87”ab,” DEXfVEE 8.46 zt 0.56 15.86 zk 0.56”,’ 18.43 zt 0.75”~’ DEX+VEE+MLT 17.99 f 0.94”,b,d 12.04 f 0.29a,b,d 7.85 rf 0.93b,d VEE 6.95 & 0.90 11.95 i 1.06” 9.94 f 0.81” MLT 22.80 zk 1.07” 22.06 zt 0.97” 22.86 i 1.42”

Values are expressed, in pg/mL, as mean i SEM. Dexamethasone (DEX; 5 mg/kg bw) was administered daily for 10 days before and 4 days after viral inoculation. Melatonin (MLT; 500 pg/kg bw) was injected S.C. daily (at 17:00) starting 3 days before and continuing through 4 days after inoculation of the virus. On the first, third and fifth day 3 mice of each group were killed for testing. “P <O.OOl when compared to control. b P CO.00 1 when compared to MLT. ‘P <O.OOl when compared to DEX. dP <O.OOl when compared to DEX+VEE.

identified as GM-CSF which promotes survival of haematopoietic progenitors by suppressing apoptosis (WILLIAMS et al., 1990). It was proposed that MLT represents a neuroendocrine regulator of IL-4 produc- tion in bone-marrow T-helper cells. IL4 may then stimulate adherent stromal cells to produce GM-CSF (MAESTRONI et al., 1994a). This neuroendocrine-cyto- kine mechanism may explain the haematopoietic rescue of MLT as well as its antitumoural and immunoenhan- cing properties.

Dexamethasone markedly reduces GM-CSF produc- tion in tumour necrosis factor alpha-stimulated fibro- blasts at both the nrotein and the RNA levels ~TOBLER et al., 1991). DEX ireatment in vitro also repressed GM- CSF expression in a human bronchial epithelial cell-line through transcriptional mechanisms (ADKINS et al., 1998), it inhibits the release of GM-CSF from human- airway smooth-muscle cells (SAUNDERS et al., 1997) and its production by human eosinophils cultured on laminin (WALSH & WARDLAW, 1997). Although our results did not evidence any change in serum levels of GM-CSF in mice treated with DEX, the significant reduction of GM- CSF (37% to 55%) detected in the animals treated with DEXSMLT, when compared to those treated with MLT, suggests that DEX treatment diminishes the effect of MLT on GM-CSF synthesis and/or release, but the mechanism of this effect remains to be elucidated.

It is interesting to note that the progressive increase in GM-CSF detected in the DEX-treated infected mice (DEX+VEE) goes together with the decrease in the survival rate. However, the progressive decrease in the levels of GM-CSF observed in the immunodepressed MLT-treated (DEXfVEEfMLT) mice from day 1 to day 5 postinoculation coincides with the increase in the survival rate of the animals. These results could be the consecmence of the effect of MLT in the oxidative metabolism. MLT’s important actions as antioxidant and free-radical scavenger may be ofprimary significance in haematopoietic tissue where oxygen-centre radicals are found in relative abundance (REITER et al., 1994).

The rise in serum levels of GM-CSF produced by the VEE viral infection on days 3 and 5 postinoculation could be due to the increment in IL-16 found in VEE virus-infected mice (BONILLA et al., 1998). In fact, it has been previously demonstrated that IL-1 induces the nroduction of GM-CSF (BAGBY. 19891.

In the present work, it was observed that the rise in GM-CSF levels caused by VEE virus infection was potentiated by DEX. However, MLT administration

on these immunodepressed infected animals diminished this effect. In fact, on the fifth day postinoculation the values of GM-CSF were lower (P <O*OO 1) than those observed in infected mice treated with DEX and similar to those detected in DEX-treated and VEE virus- infected animals.

An increase in IL-2 production in immunodepressed mice treated with MLT, with variable effects in normal young mice, has been observed (CAROLEO et al., 1994). Hypoproduction of IL-2 has been reported in several immunodeficient conditions, such as cancer (OPPEN- HEIM et al., 1987), ageing (FRASCA et al., 1986) or induced by the immunosuppressive agents cyclosporin and cyclophosphamide (SMITH, 1980). However, in this work no changes in serum IL-2 levels in any of the animal groups were detected. These results are in agreement with the findings that IL-2 production is not affected in MLT-treated and non-treated mice 3 days after the infection with VEE virus (BONILLA et al.. 1997).

This work also showed that immunodepression did not affect VEE viral replication in the brain. The lack of effect of MLT on the viral titres in the brain of immunodepressed mice contrasts with our previous work in which we found that MLT administration reduced VEE virus levels in the brain of infected immunocompetent mice on days 3,4, and 5 postinocu- lation (BONI~ et al., 1997), suggesting that the anti- viral mechanisms stimulated bv MLT in the

_I

immunocompetent mice are absent or diminished in the immunodepressed animals. It is tempting to suggest that the protection provided by MLT against oxidative damage to cell components (REITER et al., 1995) could be responsible, at least in part, for the increase in the survival rate in the immunodepressed infected mice.

Recently, it was demonstrated that mice with targeted deletions in either their interferon-alpha-beta-receptor (IFNAR-1 -/-) or interferon regulatory factor 2 (IRF-2 -/-) genes were more susceptible than control mice to VEE infection. The IFNAR-1 -/- mice exhibited accelerated VEE dissemination to serum and brain when compared to control mice. In IRF-2 --- mice inducible nitric oxide svnthase (iNOS) acne induction was com- pletely abseni following VEE &us infection. When the role of cells involved in iNOS production was evaluated, it was found that primary microglial cell cultures were highly permissive to the VEE virus infection. Besides, this infection increased the levels ofnitric oxide in resting microglial cultures, but decreased nitric oxide produc- tion in IFN-gamma-stimulated microglia. These find-

210 ERNEST0 BONILLA H-AL.

ings suggest that reactive nitrogen species play an important role in VEE virus dissemination (SCHONE- BOOM et al., 2000). In this regard, ir is interesting to note that MLT inhibits cerebellar nitric oxide svnthase activ-

I

ity (POZO etal., 1997) and it is a potent scavenger ofnitric oxide, in vitro (NODA er al., 1999). On the other hand, in cultured murine macrophages, MLT has been found to inhibit nitric oxide production due to a reduction in iNOS steadv-state mRNA levels and iNOS orotein expression <GILAD et al., 1998). We are &&&y investigating whether the antioxidative effect of MLT plays a role in protecting the VEE virus-infected mice.

Acknowledgements This work was partially supported by a grant from CON-

ICIT, Venezuela (Project G-97000638).

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Received 3 July 2000; revised 18 September 2000; accepted far publication 25 September 2000