Mouse hepatitis virus 3 (MHV3) is a natural murine viral infection that, in experimental conditions, has a resistance pattern dependent on the genetic background of the animal. BALB/c mice are susceptible and adult A/J mice are resistant to MHV3. Our previous studies have shown that resistance to MHV3 infection in mouse populations can be a consequence of a T-cell dependent mechanism, in which the production of interferon-g (IFNg) and the sensitivity of macrophages (MF) to IFNg play an essential role (Vassa˜o et al., 2000) . We have shown that the resistance of A/J mice to an MHV3 isolate that we have cloned, selected and amplified, is acquired after immunization with ultraviolet inactivated MHV3 (uvMHV3) and that IFNg activation induces an antiviral state only in MF from resistant mice, leading to a partial restriction of multiplication by significantly delaying virus replication. Recently, we have demonstrated that murine MF are capable of secreting IFNg upon a synergistic stimulation with interleukin (IL)-12 and IL-18, or an autocrine and T-cell independent MF activation mechanism (Munder et al., 1998a (Munder et al., , 2001 .
In MF, nitric oxide synthase (NOS) and arginase are inducible enzymes that act on a common substrate, arginine. As a consequence, arginine is oxidized by NOS producing citrulline and nitric oxide (NO) or is hydrolyzed by arginases producing ornithine and urea. Ornithine is in turn catabolyzed by ornithine decarboxylase (ODC) for polyamine (putrescine, spermidine and spermine) biosynthesis or ornithine amino transferase (OAT) for glutamate or proline synthesis (Fig. 1) . Difluoromethylornithine (DFMO), an analog of ornithine that inhibits ODC, has been used in the study of polyamine depletion (Wang and Wong, 2003) . Polyamines, such as spermine, play several roles in cell differentiation and proliferation (Thomas and Thomas, 2001) . Despite contradictory observations, it is generally accepted that polyamines contribute to virus maturation (Cohen, 1998) . We have recently shown that a competition between NOS and arginase for their substrate takes place in mouse bone marrow-derived MF (BMMF) and that they appear to define two alternate functional states of MF, induced by, respectively, Th1 (IFNg) and Th2 cytokines (IL-4 and IL-10) (Munder et al., 1998b (Munder et al., , 1999 . These cytokines would also differentially regulate NOS2 and arginase 1 in vivo (Hesse et al., 2001) .
NO has been considered an important molecule with protective or harmful effects on the organism and is directly involved in antiviral activities (Benencia and Courreges, 1999; Guillemard et al., 1996; Kreil and Eibl, 1995; Pereira et al., 1997; Pope et al., 1998; Saura et al., 1999; Tucker et al., 1996) . Protective effects of NO have been related to infections involving ectromelia, vaccinia and herpes simplex 1 viruses (Harris et al., 1995; Melkova and Esteban, 1995) , Coxsackie B3 virus (Lowenstein et al., 1996; Saura et al., 1999) , Epstein-Barr virus (Kawanishi, 1995) and Japanese encephalitis virus (Saxena et al., 2000 (Saxena et al., , 2001 . On the other hand, there are indications that NO may not be essential for virus clearance in vaccinia virus-infected mice (Rolph et al., 1996) or tick-borne encephalitis virus infection (Kreil and Eibl, 1996) . Although arginase induction in MF and its consequences has been poorly studied (Corraliza et al., , 1997 Modolell et al., 1995) , some reports implicate arginase in antiviral activities in MF, tumor cell cytotoxicity and immune response (Bansal and Ochoa, 2003; Mistry et al., 2001; Ochoa et al., 2001) . More recent studies indicate that arginase induction is involved in host response to microbial infections although its protective or harmful role is uncertain (Morris, 2000; Ryo et al., 2000, Wu and Morris, 1998) .
Concerning MHV3 infection, we have suggested in a previous publication that the anti-MHV3 state induced by IFNg in MF, which may contribute to resistance, was not related to arginine metabolism (Pereira et al., 1997) . Later published data indicate that resistance to MHV3 is dependent on the production of NO (Pope et al., 1998) . Besides the fact that different mechanisms may contribute to resistance, a controversial finding seems to be a defect of NO production in susceptible BALB/c mice. Nevertheless, recent studies indicate that BALB/c mice are capable of producing NO in vivo (Fabre et al., 2004; Kwon et al., 2004) . Unpublished data from our group show also that, in certain conditions, BALB/c mice are capable of producing NO in vivo, as evaluated by direct Electron Paramagnetic Resonance (EPR) spectroscopy in blood and liver. Genetic differences of the virus isolate or mouse strain used, as well as the basic mechanism considered for resistance may account for the divergent findings.
In this paper we investigate arginine metabolism associated with the autocrine pathway of IFNg activation in MF of mice resistant and susceptible to MHV3.
Mice, macrophages and virus BALB/c and A/J mice were obtained from the mouse colonies of the Instituto Butantan, Instituto de Cieˆncias Biome´dicas of the University of Sa˜o Paulo or Max-Planck Institute for Immunobiology, Freiburg, Germany. They were kept under conventional conditions and used when 4-8 weeks old. Care of animals followed the appropriate guidelines.
BMMF of mice were obtained from bone marrow cells collected from femurs by flushing of cavities with Dulbecco's modified MEM (DMEM) (Biochrom AG, Berlin, Germany) supplemented with 100 U/ml of penicillin (Sigma) and 100 mg/ml streptomycin (Sigma Chemical Co., St Louis, MO). As clearly described in another publication (Munder et al., 2001) these BMMF population are homogeneously F4/80+, MAC-1+, I-A+, B7.1+, CD16/32+ with no detectable cells expressing T, B or NK cell markers (CD3, CD4, CD8, CD19, CD5, B220, DX5, NK1.1). Cells were cultured in non-toxic Teflon bags (fluorinated ethylene propylene, Biofolie 25; Heraeus GmbH, Hanau, Germany) as described (Munder et al., 1998a) . The Teflon foils were folded and sealed with a diathermal sealing apparatus (Polystar 100 B and Polystar 401 M-RPA; Rische+Herfurth GmBH, Hamburg, Germany) to give rectangular bags, and subjected to gas sterilization. Cells (5 Â 10 4 /ml) in DMEM with 10% fetal bovine serum (FBS) (Biochrom AG), 5% horse serum (Invitrogen-Gibco Co., Carlsbad, CA), 15% L929 supernatant as a source of colony-stimulating factor, 100 U/ml penicillin, 100 mg/ml streptomycin, 1% sodium pyruvate (Invitrogen-Gibco) and 0.5% 2-mercaptothanol (Sigma) were incubated for 12 days at 37 1C in the presence of 10% CO 2 . Cells were detached by repeated careful stretching of the medium, transferred to 96-well microplates at a concentration of 2 Â 10 5 cells/100 ml and used in the experiments. Peritoneal macrophages (PMF) were prepared from peritoneal exudate cells collected by peritoneal lavage with DMEM containing 10% FBS and cultured in 96-well microplates at a concentration of 2 Â 10 5 cells/ 100ml. The cells were incubated for 2 h at 37 1C in 5% CO 2 , washed three times with DMEM after vigorous shaking to remove non adherent cells and used in the experiments. Although carefully treated, the PMF population may contain a few other cells. MHV3, originally obtained by the Institute of Virology, University Louis Pasteur, Strasbourg from Dr. J.L. Virelizier, Institut Pasteur, Paris, was cloned by limiting dilution; one plaque was selected and amplified on L929 cells to serve as the inoculum for future stocks to limit spontaneous mutations. Samples were then transferred to the Max Planck Institute of Immunobiology, Freiburg and to the Laboratory of Virus Immunology, Institute Butantan, Sa˜o Paulo. Aliquots containing 2 Â 10 5 plaque-forming units per milliliter (PFU/ml) were stored at À80 1C and used in all experiments. The stocks as well as experimental samples were always titrated by plaque assay on L929 cells as previously described (Vassa˜o et al., 2000) .
In order to study the synthesis of IFNg, BALB/c or A/J mouse PMF or BMMF cultures were stimulated with 10 ng/ml of IL-12 (R&D Systems Europe Ltd., Abingdon, UK) and/or 50 ng/ml of IL-18 (PeproTech EC Ltd., London, UK) for the indicated periods. The total cellular RNA was then extracted to evaluate the expression of the IFNg gene and the supernatants collected to quantify the level of IFNg secreted. RNA was extracted using the TriFast reagent (PEQLAB Biotechnologie GmBH, Erlangen, Germany), which briefly includes the following steps: homogenization, phase separation, RNA precipitation, RNA wash and RNA solubilization. The final RNA concentrations and degree of purity were determined by optical spectroscopy. Only highly purified preparations (OD 260 / OD 280 =1.8-2.0) were used. Reverse transcription was performed by standard procedures using Moloney murine leukemia virus reverse transcriptase (Amersham Biosciences Europe GmbH, Freiburg, Germany) and 1 ml of a dilution of the reverse transcription mixture was used for amplification by polymerase chain reaction (PCR). The primers, the fragment obtained after 35 cycles and the annealing temperature were as follows: up, 5 0 -GCT CTG AGA CAA TGA ACG CT-3 0 ; down, 5 0 -AAA GAG ATA ATC TGG CTC TGC-3 0 , 227 bp, 58 1C. The PCR products were run on a 1.5% agarose gel and stained with ethidium bromide.
IFNg secreted in the supernatants was determined by the OptEIA, mouse IFNg Set, a commercially available (BD Biosciences-Pharmingen, San Diego, USA) sandwich enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's protocol. The data are expressed in pg/ml of IFNg measured in triplicate cultures7standard deviation.
Experiments were carried out in order to evaluate, in the MF, the induction of anti-MHV3 state, mediated by exogenous IFNg or the endogenous IFNg synthesized upon IL-12/IL-18 activation. BALB/c or A/J mouse PMF or BMMF cultures were activated for 24 h with 100 U/ml of IFNg (Genentech Inc., South San Francisco, CA) or 10 ng/ml of IL-12 and/or 50 ng/ml of IL-18 and then infected with MHV3 at multiplicity of infection (moi) of 0.1. Cell supernatants were collected 18 h later and the virus titers determined by plaque assay as indicated above. The data are expressed as plaque-forming units per milliliter (PFU/ml) measured in triplicate cultures7standard deviation. In order to investigate the influence of polyamine depletion by DFMO, BALB/c or A/J mouse BMMF cultures were treated for 24 h with 10 mM DFMO (EMD Biosciences Inc., Calbiochen, San Diego, CA) and then infected with 0.1 moi of MHV3 for 18 h. Supernatants were titrated and the results expressed as described above.
To evaluate the induction of NO synthesis and arginase activity, BALB/c or A/J mouse BMMF cultures were activated for 24 h with 100 U/ml of IFNg or 10 ng/ml of IL-12 and/or 50 ng/ml of IL-18 or with 20 U/ml of IL-4 (R&D Systems Europe) and 40 U/ml of IL-10 (PeproTech EC) and then the supernatants and the cells were collected to measure, respectively, the NO synthesis and arginase activity.
NO was measured as nitrite using the Griess reagent as previously described . Briefly, culture supernatants (100 ml) were mixed with 100 ml of 1% sulfanilamide, 0.1% naphtylethylenediamine dihydrochloride (NED) and 2.5% H 3 PO 4 and absorbance was measured at 540 nm in an ELISA reader (Biochrom AG). The data are expressed in mM measured in triplicate cultures7standard deviation.
Arginase activity was measured in cell lysates as previously described (Corraliza et al., 1994) . Briefly, 10 6 cells were lysed with 0.5 ml 0.1% Triton X100 and after 30 min 0.5 ml 25 mM Tris-HCL 5 mM MnCl 2 , pH 7.4, was added. The enzyme was then activated by heating for 10 min at 56 1C. Arginine hydrolysis was carried out by incubating 25 ml of the activated lysate with 25 ml of 0.5 M arginine, pH 9.7 at 37 1C for 60 min. The reaction was stopped with 400 ml of an acidic mixture (H 2 SO 4 , H 3 PO 4 and H 2 O, 1:3:7 v/v). The urea was then measured at 540 nm after addition of 25 ml of 9% alpha-isonitrosopropiophenone (dissolved in 100% ethanol) and then heating at 100 1C for 45 min. One unit of enzyme activity is defined as the amount of enzyme which catalyses the formation of 1 mmol of urea/min. The data are expressed as mU/10 6 cells measured in triplicate cultures7standard deviation.
With the aim of investigating arginine metabolism, BALB/c or A/J mouse BMMF cultures were activated for 24 h with 20 U/ml of IL-4 or 100 U/ml of IFNg (Genentech Inc.) or 10 ng/ml of IL-12 and/or 50 ng/ml of IL-18 and then infected with 0.1 moi of MHV3 for 2 h. The cultures were then washed and incubated with arginine free DMEM containing 2% FBS and 0.1 mCi of L-(U-14 C) arginine (Amersham) per well. Microplates were then incubated for 4 h at 37 1C and freeze-thawed twice at À80 1C for cell lysis.
Arginine consumption and the synthesis of its products were evaluated by Thin-Layer Chromatography (TLC) (Sessa et al., 1990) . To identify the spots, 10 ml of a solution containing 2.5 mg/ml of citrulline, glutamate, proline, ornithine, putrescine, spermidine and spermine was added to the cell lysates. 20 ml of the samples were spotted onto TLC plates (25-Cromatoplates TLC 20 Â 20 cm, Silica Gel 60 F254) (Merck KGaA, Darmstadt, Germany) and dried for 1 h at 42 1C. They were then developed in the solvent system chloroform/ methanol/ammonium hydroxide/water 1:9:4:2 (vol/vol) and dried. Spots were developed with Ninhydin (Spray Solution Merck) by heating at 120 1C for 5 min and scraped into scintillation tubes containing 6 ml Ecoscin-tATM (National Diagnostics, Atlanta, GA). Radioactivity was determined by scintillation counting (Beckman Instruments Inc., Fullerton, CA) and the values for each compound are expressed as percentage (%) of the total radioactivity measured in triplicate cultures7standard deviation.
Mean and standard deviations (bars) were calculated. The significance levels were assessed by Student's t-test at a confidence level of po0:01
IFNc induction and MHV3 replication in BALB/c and A/J mouse macrophages upon activation with IL-12 and IL-18
Studies performed to investigate IFNg induction in activated MF are shown in Fig. 2 . Only upon a synergistic IL-12/IL-18 activation, BMMF from spf BALB/c and A/J mice were capable of synthesizing the IFNg gene as shown by RT-PCR or secrete IFNg into the supernatant culture as shown by specific ELISA assay. The treatment of BMMF from these spf mice with only IL-12 or IL-18 failed to either induce expression of the IFNg gene or secretion of IFNg during the first 48 h. BALB/c mouse MF were shown to regularly synthesize higher amounts of IFNg (3200760 pg/ml for BALB/c BMMF and 1600720 pg/ ml for A/J BMMF after 72 h of activation) and the studies showed that, in BMMF of both mouse strains, the IFNg mRNA and secreted IFNg could be detected after 24 h of IL-12/IL-18 activation. The amount of secreted IFNg present in supernatants increased consistently from 24 to 72 h (from 1500720 to 3200760 pg/ml in BALB/c BMMF and from 500710 to 1600720 pg/ml in A/J BMMF). At that time (72 h) some IFNg could be detected in cultures treated only with IL-18 (1200720 pg/ml in BALB/c BMMF and 750730 pg/ml in A/J BMMF). When PMF of non-spf BALB/c or A/J mice were used in the experiments, secreted IFNg could be detected in high amounts upon activation with IL-12 or IL-18 alone (31007200 or 38007180 pg/ml for BALB/c PMF and 15007100 or 3200760 pg/ml for A/J PMF) in addition to the synergistic IL-12/IL-18 activation (4000750 pg/ml for BALB/c PMF and 3600740 pg/ ml for A/J PMF).
Cultured macrophage populations activated with IL-12, IL-18 or exogenous IFNg were assayed for their ability to restrict MHV3 after infection. The data shown in Fig. 3 demonstrate that both BMMF and PMF from resistant A/J mice were capable of restricting MHV3 multiplication only when synergistically activated with IL-12/IL-18 (inhibition of 90.9% and 94% for PMF and BMMF, respectively) (5.570.5 Â 10 5 PFU/ml and 371.1 Â 10 5 PFU/ml for control PMF and BMMF, respectively, and 573.1 Â 10 4 PFU/ml and 370.8 Â 10 4 PFU/ml for IL-12/IL-18 activated PMF and BMMF, respectively) or with exogenous IFNg (inhibition of 92.8% and 96% for PMF and BMMF, respectively) (470.7 Â 10 4 PFU/ml and 270.4 Â 10 4 PFU/ml for IFNg activated PMF and BMMF, respectively). In contrast, no MHV3 multiplication restriction was observed in macrophages from the susceptible BALB/c mice (values from 5 to 7.5 Â 10 5 PFU/ml).
As shown in Fig. 4 the background levels of nitrites and arginase activity found in cultured BMMF of BALB/c and A/J mice were low (o4 mM). Nevertheless, high amounts of nitrites could be induced upon activation with IFNg (7075 and 5075 mM for BALB/ c and A/J BMMF, respectively) or by the synergistic treatment with IL-12/IL-18 (3576 and 2072 mM for BALB/c and A/J BMMF, respectively). In both situations higher amounts of nitrites were observed in BALB/ c BMMF and no nitrite synthesis was observed upon treatment with IL-12 or IL-18 alone or with IL-4/IL-10. The MHV3 infection was capable of further enhancing the IFNg or IL-12/IL-18 triggered-nitrite release from both BMMF populations (data not shown).
On the other hand, as also shown in Fig. 4 , arginase activity was not induced in either BALB/c or A/J BMMF upon IFNg activation or synergistic treatment with IL-12/IL-18 (o 40 mU/10 6 cells). Nevertheless high and comparable amounts of arginase activity could be induced in BMMF populations from both strains upon treatment with IL-4/IL-10 (21007400 mU/10 6 cells and 18007200 mU/10 6 cells for BALB/c and A/J BMMF, respectively). The background levels of arginase activity were higher in BALB/c BMMF than in A/J ones (1574 mU/10 6 cells and 572 mU/10 6 cells for BALB/c and A/J BMMF, respectively) and the MHV3 infection did not influence significantly the arginase activity (data not shown).
The studies of arginine consumption and synthesis of citrulline, ornithine and spermine are shown in Fig. 5 . The data show that highly enhanced arginine consumption was observed in BMMF of both BALB/c and A/J mice upon activation with exogenous IFNg (1771% or 1872% for controls and 5472% or 5573% for IFNgactivated in BALB/c and A/J BMMF, respectively) but not when the cells were treated with IL-12 and/or IL-18 (1871% for BALB/c and 1873% for A/J BMMF). In these cases, MHV3 always further enhanced the arginine consumption (3072% or 3572% for controls or 6670.1% or 7274% for IFNg-activated BALB/c and A/J BMMF, respectively). Comparable values of arginine consumption were observed in BMMF of both BALB/c and A/J mice independent of the treatment.
Comparable basal levels of citrulline were observed in BALB/c and A/J BMMF, but upon IFNg activation, it was synthesized in high amounts in BMMF of both mouse strains (3174% and 4072% in BALB/c and A/J BMMF, respectively) and could be further increased by MHV3 infection (4873% and 5574% in BALB/c and A/J BMMF, respectively). It correlated with our previous data (Fig. 4) showing that IFNg was capable of inducing NO synthesis, which was also further enhanced by MHV3 infection.
Overall polyamine synthesis was found to be lower in BALB/c BMMF than in A/J ones (6.973% and 1472% of spermine in control BALB/c and A/J BMMF, respectively), which correlated with the levels of ornithine observed (471.5% and 271.5% in BALB/ c and A/J BMMF, respectively). MHV3 infection was capable of enhancing spermine synthesis (2072% and 3273% of spermine in MHV3 infected BALB/c and A/J BMMF, respectively).
IFNg activation or synergistic treatment with IL-12/ IL-18, mainly in MHV3 infected cells, led to a decreased synthesis of spermine only in A/J BMMF (1472%, 871%, 3273% and 1272% in control, IFNg-activated, MHV3 infected and MHV3 infected/IFNgactivated A/J BMMF, respectively).
Overall putrescine and spermidine synthesis (data not shown) showed the same pattern as that of spermine ( Fig. 5 ) but at lower levels (0.6970.2% and 1.370.6% of putrescine, and 0.4970.2% and 0.7670.3% of spermidine in control BALB/c and A/J BMMF, respectively; 1.1270.6% and 1.7570.5% of putrescine, and 1.170.2% and 1.2570.4% of spermidine in MHV3 infected BALB/c and A/J BMMF, respectively).
Upon stimulation with IL-4 we could observe a high consumption of arginine in both BALB/c and A/J BMMF (9572% in both BALB/c and A/J BMMF), followed by lower synthesis of citrulline (1771% and 1371% in BALB/c and A/J BMMF, respectively) and higher synthesis of ornithine (3871% and 3672% in BALB/c and A/J BMMF, respectively) and spermine (3471% and 4471% in BALB/c and A/J BMMF, respectively) when compared to IFNg activation (Fig. 5) .
As shown in Table 1 , the inhibition of ODC activity by DFMO, which leads to a depletion of polyamines, induced a decrease in the MHV3 replication in both BALB/c (770.4 Â 10 6 to 0.870.02 Â 10 6 PFU/ml in control and DFMO-treated MF, respectively) and A/J 
MHV3, a coronavirus found endemically in mouse colonies, has been used as a model of hepatotropic virus infection. Among the mechanisms proposed for resistance of some mouse strains to experimental MHV3 infection (Fingerote et al., 1996; Lamontagne et al., 1996) , we have described a T-cell dependent one in which IFNg synthesis and MF sensitivity to IFNg play a central role. Susceptible mice would develop a fatal hepatitis by lacking either the ability to synthesize IFNg or cells capable of responding to IFNg. IFNg would exert its activity by down-regulating the expression of the main viral receptor (Vassa˜o et al., 2000) . Recently, we have also shown a novel pathway of autocrine MF activation by demonstrating that MF, upon synergistic stimulation with IL-12 and IL-18, are a potent IFNgproducing cell (Munder et al., 1998a) . BMMF are a reliable non contaminated population which can support these findings (Munder et al., 2001) . So, along with the classical or paracrine MF activation by IFNg produced by lymphoid cells, an autocrine MF activation could take place, where IFNg is produced by IL-12/IL-18-activated MF. Although their relative magnitudes are not yet known, both processes occurring in MF could play a crucial role in controlling infections.
In the present work we extended our previous observations (Vassa˜o et al., 2000) concerning MF activation by exogenous IFNg, which leads to a partial restriction of MHV3 replication only in MF of resistant mice, to the participation of the autocrine MF activation in MHV3 restriction. The data presented show that synergistic activation by IL-12/IL-18 of MF from resistant (A/J) and susceptible (BALB/c) mouse strains induced the expression of the IFNg gene ( Fig. 2A) . BMMF or PMF of both spf mouse strains secreted IFNg upon a synergistic IL-12/IL-18 activation (Fig.  2C ) and the levels of secreted IFNg increased along the time of stimulation and were higher in BALB/c MF (Figs. 2B and C) . Nevertheless, an anti-MHV3 state was observed only in A/J MF upon a synergistic activation with IL-12/IL-18 or with exogenous IFNg (Fig. 3) . As expected, in the PMF of non-spf animals, IFNg synthesis could be observed also among MF stimulated only with IL-12 or IL-18. Taken together, these results demonstrate that autocrine MF activation takes place in both BALB/c and A/J MF and, as shown for paracrine IFNg MF activation, only A/J MF respond with the expression of an anti-MHV3 state. Both BMMF and PMF behave similarly regarding IFNg synthesis and anti-MHV3 state. Among non-spf mice, which better mirror the natural state of mice concerning this endemic virus infection, their PMF synthesize high levels of IFNg upon treatment with only IL-12 or IL-18 possibly due to their already immunologically primed state or to the presence of other cells. The evidence that, after 72 h, the BMMF from spf mice synthesized some IFNg upon stimulation of only IL-18, may rely on the autocrine pathway. The contribution of each, the paracrine, T-cell dependent, and the autocrine, T-cell independent, mechanism of MF activation during MHV3 infection remains to be determined. It is conceivable that autocrine-IFNg synthesis participates in early stages of infection, prior to specific antigen recognition by lymphoid cells, not only by enhancing the MF ability to restrict the agent's infectivity but also by enhancing the MF ability to present antigens. By this way, autocrine MF activation would represent a link between the innate and acquired immune response (Frucht et al., 2001) .
One of the main features occurring during MF activation involves arginine metabolism and the synthesis of key molecules such as NO on one side and arginase, which may lead to the formation of polyamines, on the other. NO is a highly reactive molecule produced from a guanidine-nitrogen of arginine in a reaction catalyzed by NOS. NO is synthesized at high levels by MF upon stimulation by Th1 cytokines (Hesse et al., 2001; Munder et al., 1999) and has been implicated in host defense (Guillemard et al., 1996; Harris et al., 1995; Kawanishi 1995 Melkova and Esteban, 1995; Tucker et al., 1996) . Arginase, an enzyme induced in MF upon stimulation by Th2 cytokines (Hesse et al., 2001; Munder et al., 1999) , drives the synthesis of ornithine, which is catalyzed by ODC to form polyamines (putrescine, spermidine and spermine). DFMO, an analog of ornithine, can irreversibly inhibit ODC causing polyamine depletion. Arginase has been implicated in virus inhibition and may potentially have an antimicrobial activity OR play an antimicrobial role Modolell et al., 1995) . Polyamines, such as spermine, play several roles in cell differentiation and proliferation and may contribute to virus maturation (Cohen, 1998; Thomas and Thomas, 2001; Wang and Wong, 2003) . Although data have shown that polyamine depletion reduces cytomegalovirus or herpes simplex virus 2 infectivity, contradictory reports are found in the literature (Cohen, 1998) .
Our data show that after activation with IL-12/IL-18 or IFNg, BALB/c and A/J BMMF released NO (Fig. 4A) . Arginase was induced only when IL-4/IL-10 was used for activation (Fig. 4B ). Higher arginine consumption was observed in BMMF from both strains upon IFNg or IL-4 activation and, as expected, IFNg activation led to higher citrulline synthesis and IL-4 activation to higher ornithine synthesis (Figs. 1, 5B and 5C). Both arginine consumption and spermine synthesis increased during MHV3 infection. Although IL-12/IL-18 activation of both BALB/c and A/J BMMF led to NO synthesis (Fig. 4A) , a higher consumption of arginine was not observed in this situation (Fig. 5A ). IL-12/IL-18 or IFNg activation, mainly in MHV3 infected cells, led to a decreased synthesis of spermine only in A/J BMMF (Fig. 5D ). The putrescine and spermidine pattern levels followed that of spermine but at lower levels (data not shown). Intracellular polyamines were shown to be important during MHV3 replication since, in DFMO-treated BMMF, we detected a significant decrease in MHV3 titer (Table 1) .
Our data of arginine metabolism in MF (Fig. 5) are consistent with the notion that the newly established autocrine MF activation leads to IFNg synthesis which in turn induces NO synthesis ( Fig. 2 and 4) . The lack of arginase induction indicates the Th1 behavior of this type of autocrine activation. The fact that upon exposure to IL-12/IL-18, which induces endogenous IFNg (Fig. 2) and NO (Fig. 4) , we failed to observe the expected arginine consumption and citrulline synthesis, clearly observed when exogenous IFNg was employed (Figs. 5A and B), may rely on inherent quantitative and kinetic characteristics of the process.
Regarding the mechanism involved in resistance/ susceptibility of mice to MHV3 infection, our previous (Pereira et al., 1997; Vassa˜o et al., 2000) and present data obtained with BALB/c and A/J MF, clearly link IFNg synthesis, anti-MHV3 state induction and poly-amine inhibition in A/J MF (Fig. 6A ). In these MF, polyamines were induced during MHV3 infection and significantly inhibited upon activation by IFNg or IL-12/IL-18. Supported by the data showing a decreased MHV3 replication in DFMO treated MF (Table 1) , the polyamine modulation by IFNg correlates with the anti-MHV3 state observed only among A/J MF. Nevertheless, the molecular basis of a mechanism underlying the influence of IFNg on polyamine synthesis remains unclear.
Our findings show that both autocrine and paracrine processes of MF activation may participate in the inhibition of MHV3 replication and that the involvement of arginine metabolism in resistance to MHV3 may rely on the decreased synthesis of polyamines induced by IFNg (Figs. 5D and 6)We can speculate that polyamines, besides their effects on cell differentiation, may participate directly in MHV3 maturation.
Altogether these data show the relevance of IFNg, from autocrine or paracrine pathways, and suggest how arginine metabolism may be involved in the modulation of MHV3 replication in MF of a resistant mouse strain.