Abstract
We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-γ), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-γ release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-γ production was found in uninfected mice administered IL-18. Although IFN-γ has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor NG-monomethyl-l-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-γ produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.
Interleukin-18 (IL-18) is a newly cloned murine and human cytokine (28, 36) previously called gamma interferon (IFN-γ)-inducing factor. It is synthesized by activated macrophages and has a structural relationship to the IL-1 family (5). Precursor IL-18 is processed by IL-1β-converting enzyme and is cleaved into mature IL-18 (11). IL-18 induces IFN-γ production by murine helper T cells and NK cells and stimulates T-cell proliferation and NK activation (18, 28). Moreover, IL-18 augments the Fas ligand-mediated cytotoxic activity of the Th1 clone and the NK cell clone (8, 35). Thus, IL-18 shares some biological activities with IL-12, although no significant homology between the two cytokines has been detected at the protein level (34). Furthermore, treatment with IL-12 and IL-18 has a synergistic effect on IFN-γ production (2, 14, 38, 40).
According to a review by Nash (27), not only nonspecific or innate immunity, such as that from IFN, NK cells, or macrophages, but also specific or adaptive immunity is important in protection against herpesvirus infection. Herpes simplex virus is known to be an IFN inducer (13). IFN is produced at an early stage of virus infection. In addition to the direct inhibition of viral replication, it enhances the efficiency of the adaptive (specific) immune response by stimulating increased expression of major histocompatibility complex class I and II or by activating macrophages and NK cells. In protection from infection by herpesviruses, especially cytomegalovirus, NK cells have been major effector cells because of the correlation of increased susceptibility to cytomegalovirus infection with the absence or reduction of NK cell activity, as seen in Chediak-Higashi syndrome patients and beige mice (27). Upon target cell disruption, NK and cytotoxic T cells share not only the perforin but also the Fas ligand as an effector molecule (4, 20, 37). Recently, nitric oxide (NO) was reported to be involved in host defense against bacteria, fungi, parasites, and viruses (10, 16, 19, 39). NO produced by herpes simplex virus type 1 (HSV-1)-infected macrophages is reported to inhibit viral replication (7). CD4+ T cells, macrophages, IFN-γ, and tumor necrosis factor (TNF) are important in adaptive immunity against HSV-1 infection. The Th2 response exacerbates HSV-1-induced disease (25).
Recently a protective role of IL-18 was reported in microbial infections (6, 17). Here, we demonstrate that IL-18 treatment protects mice from acute viral infection via both IFN-γ-dependent and -independent pathways. Although IFN-γ has the ability to induce NO production by a variety of cells, including macrophages (9), it is not likely to be important, at least in the inhibition of HSV-1 proliferation at the injection site of IL-18-treated mice. Furthermore, the protective effect of IL-18 on HSV-1 infection also does not seem to require complete NK cell activity in our experimental system, whereas our colleagues have already reported that deletion of NK cells by administration of anti-asialo GM1 antibody resulted in lowering of the improved survival rate of tumor-bearing mice treated with IL-18 (23).
MATERIALS AND METHODS
Mice.
Female BALB/c, BALB/c nude (nu/nu), BALB/c (nu/+), and BALB/c SCID mice were purchased from Japan Charles River Inc. (Kanagawa, Japan), and female BALB/c IFN-γ knockout (IFN-γ −/−) mice were from The Jackson Laboratory (Bar Harbor, Maine). These mice were housed in a specific-pathogen-free environment and used at ages of 8 to 10 weeks.
Infection of mice.
The Miyama strain of HSV-1 was propagated in human colon tumor WiDr cells or monkey kidney Vero cells and titrated by the plaque-forming assay on Vero cells. For survival experiments, all of the strains of mice used were infected intraperitoneally (i.p.) with a dose of 104 PFU per mouse 2 h after administration of the vehicle or IL-18. Their survival was checked every day until 3 weeks after infection. HSV-1 at 104 PFU was equivalent to 10 times the 80% lethal dose for BALB/c mice.
Reagents.
Recombinant murine IL-18 is a product of Hayashibara Biochemical Laboratories Inc. (Okayama, Japan) and was obtained by expression of murine IL-18 cDNA in Escherichia coli and then purification by chromatography as described previously (28). The level of endotoxin in the sample was less than 9 ng/mg of IL-18. IL-18 was diluted with saline supplemented with 0.1% mouse serum albumin and was given i.p. at 1 μg/mouse in a volume of 0.1 ml daily from day 2 before infection to day 1 after infection. Control mice were injected with the diluent. To inhibit NK cell activity in vivo, rabbit anti-asialo GM1 antibody (Wako, Osaka, Japan) was injected i.p. at a pretitrated dose that depleted IL-18-induced antitumor NK cell activity in BALB/c mice. The antibody and normal rabbit serum (NRS; used as a control) were given to mice 3 days and 1 day before infection and 1 day after infection and every third day thereafter until all of the mice treated with NRS died. To inhibit NO synthesis in vivo, the mice were injected i.p. at 2 mg/mouse with NG-monomethyl-l-arginine (l-NMMA; Calbiochem-Novabiochem Co., La Jolla, Calif.) dissolved in 0.2 ml of phosphate-buffered saline (pH 7.2) daily from day 3 before infection to the day of death of all infected control mice, which was the same amount as the control, NG-monomethyl-d-arginine (d-NMMA; Calbiochem-Novabiochem Co.) (1, 32). The level of endotoxin in all of the injections was less than 20 pg/ml (Limulus Amebocyte Lysate QCL-1000; BioWhittaker, Walkersville, Md.).
Tissue sampling and culture of peritoneal cells.
Blood was collected from mice by cardiac puncture, and the serum was stored at −40°C until virus assay or enzyme-linked immunosorbent assay (ELISA) for cytokines. Peritoneal cells were harvested by washing of the peritoneal cavity with 4 ml of ice-cold RPMI 1640 medium (supplemented with 2% fetal bovine serum [FBS] and 60-μg/ml kanamycin per mouse. After centrifugation, the peritoneal washing fluid (PWF) supernatants were stored at −40°C until virus assay or cytokine ELISA. In some experiments, the peritoneal cells in the PWF were washed twice and then suspended at 5 × 106/ml in RPMI 1640 medium supplemented with 2% FBS. A 100-μl suspension was added to each well of a 96-well microplate and incubated with or without IL-18 (1 ng/ml) at 37°C for 24 h in an atmosphere of 5% CO2. The culture supernatants harvested were stored at −40°C until assayed for cytokine or NO. In some experiments, spleens were obtained from mice to prepare suspensions.
Measurement of antiviral activity.
Antiviral activity was measured by using a cytopathic effect reduction assay with vesicular stomatitis virus (VSV) as the challenge virus and mouse L929 cells. In brief, monolayers of L929 cells were incubated in medium (Eagle’s minimum essential medium supplemented with 2% FBS) containing the samples at 37°C for 1 day after UV irradiation of the samples to inactivate infectious HSV-1. After removal of the medium, L929 cells were inoculated with an appropriate titer of VSV and then incubated at 37°C for 1 day. Cell viability was examined by incorporation of neutral red. Natural mouse IFN-α (mIFN-α), a product of our laboratory, was used as the positive control, and the antiviral activity of the sample was expressed as the mIFN-α titer in international units.
Cytokine assay.
Mouse IFN-γ and mouse TNF-α were measured by using a specific ELISA. Briefly, microtiter plates coated with rabbit polyclonal anti-mouse IFN-γ antibody prepared in our laboratory or rat monoclonal anti-mouse TNF-α antibody (PM-18131D; PharMingen, San Diego, Calif.) were incubated with sample dilutions. After washing, biotinylated rat monoclonal anti-mouse IFN-γ antibody (PM-18112D; PharMingen) or rabbit polyclonal anti-mouse TNF-α antibody (PM-18352D; PharMingen) was added. After washing, horseradish peroxidase-conjugated streptavidin (43-4323; Zymed Laboratories Inc., South San Francisco, Calif.) was added. The plates were developed by using o-phenylenediamine (Wako) in citrate buffer. The reaction was stopped with 2 N H2SO4, and absorbance was read at 490 to 630 nm by using a microplate reader. Recombinant mouse IFN-γ, a product of our laboratory, and recombinant mouse TNF-α (PM-19321T; PharMingen) were used as standards. Mouse IL-18 was measured by using a rat monoclonal anti-mouse IL-18 antibody obtained from animals immunized with recombinant mouse IL-18 in our laboratory. Recombinant mouse IL-18 was used as the standard.
Analysis of asialo GM1+ cells.
Peritoneal cells or splenocytes from mice injected with NRS or anti-asialo GM1 antibody were suspended at 5 × 106/ml in RPMI 1640 medium supplemented with 10% FBS after being washed twice. NRS or anti-asialo GM1 antibody was added to a 100-μl cell suspension at a final dilution of 1:150, and the suspensions were then incubated at 37°C for 30 min. Guinea pig serum (Inter-Cell Immunologies, Inc., Hopeville, Mass.) was then added as complement for a final dilution of 1:40, and the suspensions were then incubated at 37°C for 30 min. The killing of asialo GM1+ cells by antibody and complement was determined by trypan blue dye exclusion and by counting more than 400 cells. The percentage of asialo GM1+ cells was calculated by subtracting the percent cytotoxicity of NRS plus complement from that of antibody plus complement. Cytotoxicity by complement alone was less than 3.0%.
NO assay.
For measurement of nitrite (NO2−) in the cell culture, the supernatant samples were mixed with equal volumes of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid), and then the optical density at 540 nm was measured by spectrophotometry. Sodium nitrite was used as a standard for each experiment. For measurement of nitrate (NO3−) in the PWF or serum, the PWF samples were prepared by using ice-cold phosphate-buffered saline to wash the peritoneal cavities of the mice, after which the samples of PWF or serum were deproteinized by filtration through a Millipore Ultrafree-MC (nominal molecular weight limit; 10,000) filter unit (Millipore Corp., Bedford, Mass.). The sample nitrate was then measured by using an NO assay kit (Cayman Chemical Company, Ann Arbor, Mich.).
Statistical analysis.
The survival data were analyzed by a generalized Wilcoxon test. Treatment group differences in viral titer and cytokine amount were examined by using the Student t test.
RESULTS
Effect of exogenous IL-18 on survival of HSV-1-infected mice.
Preliminary studies indicated that 4 days of treatment (from day 2 before infection to day 1 after infection) with 1 μg of IL-18 per mouse daily was sufficient to protect BALB/c mice from acute lethal HSV-1 infection, but 4 days of treatment with 0.01 or 0.1 μg of IL-18 per mouse daily was not. The lack of IL-18-induced toxicity reflected on IL-18-induced resistance to HSV-1, since there were no ill effects in uninfected mice treated with daily doses of IL-18 of 0.01 to 5 μg per mouse for 4 or more days (data not shown).
Treatment with 1 μg of IL-18 was significantly effective for protection of BALB/c, BALB/c nude, and BALB/c SCID mice from HSV-1. Since BALB/c nude and SCID mice have a deficiency in T-cell function and in T- and B-cell functions, respectively, the protective effect of IL-18 against HSV-1 infection suggested the involvement of innate immunity (Fig. 1). There was no difference in protection between IL-18-treated BALB/c homozygous nude (nu/nu) mice and their heterozygous (nu/+) littermates, used to evaluate T-cell-dependent immunity, revealing the importance of innate immunity over adaptive immunity.
FIG. 1.
Effect of IL-18 treatment on survival of BALB/c (+/+), BALB/c SCID, and BALB/c homozygous nude (nu/nu) and heterozygous (nu/+) mice infected i.p. with 104 PFU of HSV-1. IL-18 was administered i.p. at 1 μg per mouse daily from day 2 before infection to day 1 after infection. Open and closed circles indicate groups of mice (n = 5 per group) treated with the vehicle or IL-18 respectively. The data is representative of two or more independent experiments with similar results. ∗, P < 0.05.
Effect of IL-18 on HSV-1 replication and cytokine production in BALB/c mice.
Since IL-18 treatment improved the survival of HSV-1-infected BALB/c mice, we examined HSV-1 replication and cytokine production in vehicle- and IL-18-treated BALB/c mice. On day 1 after infection, HSV-1 was detected in the PWF of vehicle-treated mice but was almost undetectable in that of IL-18-treated mice (Fig. 2A), whereas infectious virus was undetectable 2 to 3 h after infection. Infectious virus was not found at the injection site more than 1 day after infection, nor was viremia seen during infection (data not shown). The antiviral activity and cytokine levels in the PWF and serum were then measured. Although both levels were almost undetectable in the PWF and serum of both control and IL-18-treated mice immediately before HSV-1 inoculation, the antiviral activity was significantly higher in the PWF of the control mice than in that of the IL-18-treated mice on day 1 of infection (Table 1). On day 1 after infection, IFN-γ was also detected in the PWF of both the control and IL-18-treated mice, but the difference in titers was not significant. No significant differences in antiviral activity and IFN-γ titer were detected between the sera of control and IL-18-treated mice 1 day after infection. Neither group of mice had any appreciable IFN-γ, TNF-α, or antiviral activity in the PWF or serum on days 3 and 5 of infection (data not shown). Circulating IL-18 was detected in IL-18-treated mice (e.g., 5 days after infection, <5.2 ± 2.9 ng/ml, [mean ± standard deviation, n = 5) but not in control mice during infection (<0.25 ± 0 ng/ml, n = 5). Furthermore, no neutralizing anti-HSV-1 antibody was detected in the PWF (1:<4, n = 5) or the serum pooled from five mice (1:<24) of both the control and IL-18-treated groups until the moribund stage of the disease in the control mice.
FIG. 2.
Virus titers of PWF from IL-18-treated mice inoculated i.p. with 104 PFU of HSV-1, determined 26 h after infection. IL-18 was administered i.p. at 1 μg per mouse daily from day 2 before infection to day 1 after infection. The NO synthase inhibitor l-NMMA or the control d-NMMA was injected i.p. on days 3, 2, 1, and 0 before infection and day 1 after infection. The final injection of l- or d-NMMA was given 2 h before IL-18 administration. The PWF of all groups was prepared 2 h after the final IL-18 injection. Open and closed circles indicate undetectable and detectable values for each mouse, respectively. Panels: A, vehicle- or IL-18-treated BALB/c mice; B, vehicle- or IL-18-treated BALB/c IFN-γ −/− mice; C, vehicle- or IL-18-treated BALB/c mice injected with d- or l-NMMA. ∗, ∗∗, P < 0.05 and P < 0.01, respectively.
TABLE 1.
Profiles of antiviral cytokines in PWF and serum obtained from BALB/c IFN-γ +/+ and IFN-γ −/− mice 26 h after i.p. inoculation with 104 PFU of HSV-1
| Exptl group | IL-18 treatmenta | PWF
|
Serum
|
||||
|---|---|---|---|---|---|---|---|
| Antiviral activityb (mIFN-α IU/ml) | IFN-γc (101 pg/ml) | TNF-αc (pg/ml) | Antiviral activity (mIFN-α IU/ml) | IFN-γ (101 pg/ml) | TNF-α (pg/ml) | ||
| IFN-γ +/+ | − | 31.8 ± 18.4d | 90 ± 45 | <44 ± 27 | <57.8 ± 42.2 | <30 ± 0 | <79 ± 0 |
| + | <0.4 ± 0.0e | 40 ± 15 | <66 ± 48 | <10.0 ± 0.0 | <45 ± 15 | <79 ± 0 | |
| IFN-γ −/− | − | 36.2 ± 16.9 | NDf | <31 ± 0 | 45.6 ± 35.5 | ND | <79 ± 0 |
| + | 10.4 ± 4.3e | ND | <31 ± 0 | <5.0 ± 0.0e | ND | <79 ± 0 | |
On days 2 and 1 before infection and days 0 and 1 of infection, 1 μg of IL-18 was injected i.p. into each mouse (n = 5 per group), and the mice were sacrificed to prepare PWF and serum 2 h after the final injection with the vehicle or IL-18.
Antiviral activity was measured by using VSV and mouse L929 cells and is expressed as the mIFN-α titer.
Measured by ELISA.
Mean ± standard deviation.
Significantly decreased values compared to those of vehicle-treated mice (P < 0.01).
ND, not done.
Effect of IL-18 on HSV-1 infection in BALB/c IFN-γ −/− mice.
The IFN-γ titer in the PWF of IL-18-treated mice was not higher than that of vehicle-treated mice on day 1 after infection, even though IFN-γ has been reported to be produced by NK and B cells after stimulation with IL-18 in vitro (14, 38). To determine whether IFN-γ is involved in IL-18-induced immunity to HSV-1 infection or not, we examined the antiviral effect of IL-18 by using BALB/c IFN-γ −/− mice. IL-18 treatment failed to protect the mice significantly but did prolong the mean survival time slightly (control, 5.8 days; IL-18, 9.0 days, n = 5) (Fig. 3). IFN-γ deficiency restored the early HSV-1 growth at the injection site in IL-18-treated mice, yet HSV-1 titers in the PWF of IL-18-treated IFN-γ −/− mice were sixfold lower than those of control IFN-γ −/− mice (Fig. 2B). On day 1 after infection, however, antiviral activity was higher in the PWF of vehicle-treated IFN-γ −/− mice than in that of IL-18-treated IFN-γ −/− mice, and TNF-α was undetectable in the PWF of both vehicle- and IL-18-treated IFN-γ −/− mice (Table 1). These results indicate that not only IFN-γ-dependent but also IFN-γ-independent pathways play an important role in IL-18-promoted protection against HSV-1 infection.
FIG. 3.
Effect of IL-18 treatment on survival of BALB/c IFN-γ +/+ and IFN-γ −/− mice inoculated i.p. with 104 PFU of HSV-1. IL-18 was administered i.p. at 1 μg per mouse daily from day 2 before infection to day 1 after infection. Open and closed circles indicate groups of vehicle and IL-18-treated mice (n = 5 per group), respectively. The data is representative of two experiments with similar results. ∗, P < 0.05.
Effect of anti-asialo GM1 treatment on HSV-1 infection in IL-18-treated BALB/c mice.
Although IFN-γ was involved in the protective effect of IL-18 against HSV-1 infection, there remains an IFN-γ-independent pathway(s) which inhibited early HSV-1 replication and prolonged the mean survival time in IL-18-treated IFN-γ −/− mice. IL-18 has been reported to enhance NK cell killing in vivo and in vitro (22, 28), and a protective role of NK cells through perforin-dependent cytolysis has been observed in herpesvirus infections (27, 32). Additionally, it has been reported that mouse peritoneal exudate cells inhibit microbial growth via IFN-γ release by NK cells and subsequent NO production in the presence of IL-12 plus IL-18 in vitro (40). To determine whether such NK cell activities are involved in the IL-18-promoted protection against HSV-1 infection, we examined the effect of anti-asialo GM1 treatment on HSV-1 infection in IL-18-treated mice. Treatment of mice with IL-18 augmented splenic NK cell killing of Yac-1 cells (22). However, injection of anti-asialo GM1 antibody into normal or IL-18-treated mice resulted in the complete loss of such NK activity, whereas NRS injection had no such effect (31). Treatment with anti-asialo GM1 antibody or NRS had no influence on the survival of HSV-1-infected mice administered IL-18 or the vehicle (Fig. 4A and B), in contrast to tumor-bearing mice (23). On day 1 after infection, a reduction in the asialo GM1+ cell population was observed in the peritoneal cells and splenocytes of antibody-treated mice (Table 2). Both the NRS and antibody treatment groups of mice had almost the same level of virus production after vehicle administration, but after IL-18 administration, both groups had undetectable levels of virus in the peritoneal cavity on day 1 of infection (Table 3). Antiviral activity was detected in the PWF and serum of both the NRS and antibody treatment groups after vehicle administration but not in those of either group after IL-18 administration. No appreciable IFN-γ production was found in the PWF or serum of any treatment group except the NRS-plus-IL-18 group. Additionally, neither group of mice had any appreciable TNF-α in the PWF (<31 ± 0 pg/ml) or serum (<79 ± 0 pg/ml). Antiviral activity, IFN-γ, and TNF-α were all undetectable in the PWF and serum of vehicle- or IL-18-treated mice administered NRS or antibody when HSV-1 was not injected into the mice. Therefore, the results suggested that treatment with NRS or anti-asialo GM1 antibody had no marked influence on virus replication or the induction of antiviral activity or of TNF-α production. However, it did modify IFN-γ production in mice administered the vehicle or IL-18 1 day after infection. It also suggested that complete NK cell activity is not required for the anti-HSV-1 effect of IL-18.
FIG. 4.
Effect of treatment with anti-asialo GM1 antibody and l-NMMA on survival of IL-18-treated BALB/c mice infected i.p. with 104 PFU of HSV-1. IL-18 was administered i.p. at 1 μg per mouse daily from day 2 before infection to day 1 after infection. NRS or antibody was injected i.p. on days 3 and 1 before infection and day 1 after infection and every 3 days thereafter. d- or l-NMMA was injected i.p. daily from day 3 before infection to the day of death of all l-NMMA-treated mice injected with the vehicle. Open and closed circles indicate groups of vehicle and IL-18-treated mice (n = 5 per group), respectively. The data is representative of two experiments with similar results. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively.
TABLE 2.
Asialo GM1+ cells in vehicle- and IL-18-administered BALB/c mice treated with NRS or anti-asialo GM1 antibody 26 h after i.p. inoculation with 104 PFU of HSV-1
| In vivo treatment | Mean % asialo GM1+a
|
|
|---|---|---|
| Peritoneal cells | Splenocytes | |
| NRSb + vehiclec | 5.1 | 14.4 |
| AGMd + vehicle | −0.3 | 1.6 |
| NRS + IL-18 | 17.4 | 9.7 |
| AGM + IL-18 | 2.3 | −3.0 |
A cytotoxicity test using NRS or anti-asialo GM1 antibody plus complement was conducted to determine the percentage of asialo GM1+ cells.
NRS or anti-asialo GM1 antibody was injected i.p. on days 3 and 1 before infection.
On days 2 and 1 before infection and days 0 and 1 of infection, 1 μg of IL-18 was injected i.p. into each mouse (n = 2 per group), and the mice were sacrificed to prepare peritoneal cells and splenocytes 2 h after a final injection with the vehicle or IL-18.
AGM, anti-asialo GM1 antibody.
TABLE 3.
HSV-1 and cytokine titers in PWF and serum from vehicle- and IL-18-administered BALB/c mice treated with NRS or anti-asialo GM1 antibody 26 h after i.p. inoculation with 104 PFU of HSV-1
| In vivo treatment | PWF
|
Serum
|
|||
|---|---|---|---|---|---|
| HSV-1a (log10 PFU/ml) | Antiviral activityb (mIFN-α IU/ml) | IFN-γc (101 pg/ml) | Antiviral activity (mIFN-α IU/ml) | IFN-γ (101 pg/ml) | |
| NRSd + vehiclee | 1.7 ± 0.2f | 17.6 ± 6.0 | <6 ± 0 | 29.7 ± 13.4 | <30 ± 0 |
| AGMh + vehicle | 1.8 ± 0.2 | 14.7 ± 5.5 | <6 ± 0 | 18.3 ± 8.5 | <30 ± 0 |
| NRS + IL-18 | <0.7 ± 0.0 | <1.0 ± 0.0 | 48 ± 20g | <10.0 ± 0.0 | <30 ± 0 |
| AGM + IL-18 | <0.7 ± 0.0 | <1.0 ± 0.0 | <6 ± 0 | <10.0 ± 0.0 | <52 ± 28 |
HSV-1 was titrated by plaque assay with Vero cells.
Antiviral activity was measured by using VSV and mouse L929 cells and is expressed as the mIFN-α titer.
Measured by ELISA.
NRS or anti-asialo GM1 antibody was injected i.p. on days 3 and 1 before infection.
On days 2 and 1 before infection and days 0 and 1 of infection, 1 μg of IL-18 was injected i.p. into each mouse (n = 3 per group), and the mice were sacrificed to prepare PWF and serum 2 h after the final injection with the vehicle or IL-18.
Mean ± standard deviation.
Significantly increased values compared to those of mice treated with anti-asialo GM1 antibody plus IL-18 (P < 0.01).
AGM, anti-asialo GM1 antibody.
Effect of IL-18 treatment in vivo and in vitro on IFN-γ production by peritoneal cells from uninfected BALB/c mice.
Heightened IFN-γ production was not found in IL-18-treated mice versus control mice in HSV-1 infection, and no appreciable IFN-γ production was observed in IL-18-treated uninfected mice, although IFN-γ was the key cytokine for the IL-18-promoted protection against HSV-1 infection. Since IL-18 has already been reported to induce or augment IFN-γ release by NK and B cells in vitro (14, 38), we examined the effect of IL-18 on IFN-γ synthesis by peritoneal cells from uninfected mice to assess the possibility that an undetectably low level of IFN-γ is produced in the peritoneal cavities of mice injected with IL-18 before HSV-1 infection.
As shown in Table 4, IL-18 enhanced in vitro IFN-γ production by peritoneal cells from mice treated with the vehicle but not that of peritoneal cells from mice treated with IL-18. Furthermore, IL-18 augmented IFN-γ release by peritoneal cells from vehicle- and IL-18-treated mice injected with NRS or anti-asialo GM1 antibody. In the peritoneal cells from antibody-treated mice, asialo GM1+ cells were almost totally depleted (data not shown). Apparently, anti-asialo GM1 treatment in vivo led the peritoneal cells from uninfected mice administered the vehicle or IL-18 into weakened IFN-γ production upon in vitro stimulation with IL-18. The results suggested that exogenous IL-18 can induce or augment IFN-γ production by peritoneal cells without infection and that not only NK cells but also other cells can participate in it in response to IL-18 stimulation.
TABLE 4.
Effect of IL-18 treatment in vitro on IFN-γ production by peritoneal cells from uninfected BALB/c mice treated with or without anti-asialo GM1 antibody
| Expt and in vivo treatment | IFN-γ (ng/ml)a
|
Increase (fold)b | |
|---|---|---|---|
| IL-18(−) | IL-18(+) | ||
| 1 | |||
| Vehiclec | 1.9 | 7.0 | 3.7 |
| IL-18 | 0.7 | 0.7 | 1.0 |
| 2 | |||
| NRSd + vehicle | 1.1 | 11.5 | 10.5 |
| NRS + IL-18 | 2.9 | 6.5 | 2.2 |
| AGMe + vehicle | 1.9 | 5.0 | 2.6 |
| AGM + IL-18 | <0.2 | 0.6 | >3.0 |
Peritoneal cells were incubated in the presence or absence of IL-18 (1 ng/ml), and the culture supernatants were harvested 24 h later. IFN-γ in the supernatant pooled from duplicate or triplicate samples was measured by ELISA.
IFN-γ amount after IL-18(+) treatment/IFN-γ amount after IL-18(−) treatment.
The vehicle or IL-18 (1 μg per mouse a day) was administered i.p. to mice for 3 days, and peritoneal cells were prepared 1 day after the last administration.
NRS or anti-asialo GM1 antibody was injected i.p. into mice twice, 1 day each before and after the first administration of the vehicle or IL-18.
AGM, anti-asialo GM1 antibody.
Effect of l-NMMA treatment on HSV-1 infection in IL-18-treated BALB/c mice.
One of the ways IFN-γ can inhibit virus replication is by inducing NO production, which has been shown to inhibit ectromelia virus, vaccinia virus, and HSV-1 replication in vivo and in vitro (12, 16). To determine whether more NO is produced in IL-18-treated mice than in control mice, we measured the nitrate in the PWF and serum of HSV-1-infected mice treated with the vehicle or IL-18. Nitrate was undetectable in the PWF of both groups (<1.0 μM). On days 1 and 3 after infection, the serum nitrate levels were lower in IL-18-treated mice but higher in control mice than in normal mice (Fig. 5). Furthermore, in the in vitro experiments, IL-18 augmented nitrite production by peritoneal cells from uninfected mice treated with the vehicle but not by peritoneal cells from uninfected mice treated with IL-18: vehicle treatment, IL-18(−) at 1.0 μM versus IL-18(+) at 19.0 μM; IL-18 treatment, IL-18(−) at 1.0 μM versus IL-18(+) at 1.0 μM (pooled supernatants from two or three samples). Additionally, nitrite production by peritoneal cells from mice 1 day after 3 days of treatment with the vehicle and infection was higher than with IL-18 (3.0 versus 1.0 μM). The results indicated that NO synthesis is weaker in IL-18-treated mice than in control mice in the early stage of infection.
FIG. 5.
Serum nitrate levels in vehicle- or IL-18-treated BALB/c mice during i.p. infection with 104 PFU of HSV-1. Mice were i.p. administered the vehicle or IL-18 (1 μg per mouse) daily from day 2 before infection to day 1 after infection. Nitrate levels in pooled serum are shown (n = 5).
Further, to determine whether HSV-1 is regulated by IFN-γ via NO production, we examined the effect of an NO synthase inhibitor on the survival of infected mice treated with IL-18. Treatment with l-NMMA prolonged the mean survival time of vehicle-treated infected mice (d-NMMA plus vehicle, 5.8 days; l-NMMA plus vehicle, 9.4 days) but diminished the improved survival of IL-18-treated infected mice (d-NMMA plus IL-18, >16.0 days; l-NMMA plus IL-18, >11.8 days), revealing a diminution of the statistically significant difference in the survival rate (Fig. 4C and D). Therefore, we measured the HSV-1 titer and cytokine concentration in the PWF and serum 1 day after infection. Both the d- and l-NMMA treatment groups had almost undetectable HSV-1 levels after IL-18 administration, but the same groups had more virus, the titers of which were not significantly different, in the peritoneal cavity after vehicle administration on day 1 of infection (Fig. 2C). As shown in Table 5, induction of antiviral activity and that of IFN-γ were seen in the PWF of both the d- and l-NMMA treatment groups after vehicle administration but not in that of either treatment group after IL-18 administration. No TNF-α was detected in the PWF of either treatment group administered the vehicle or IL-18 (<31 ± 0 pg/ml). Furthermore, neither virus nor cytokine was detectable in the serum of d- or l-NMMA-treated mice administered the vehicle or IL-18. In addition, 1 day after infection and treatment with d- or l-NMMA, the nitrate concentration of the serum pooled from mice (n = 3 per group) treated with l-NMMA was lower than that of the d-NMMA treatment group: d-NMMA plus vehicle, 4.0 μM versus l-NMMA plus vehicle, 1.5 μM; d-NMMA plus IL-18, 2.5 μM versus l-NMMA plus IL-18, 1.5 μM. The results suggested that l-NMMA treatment did not have any influence on the replication of HSV-1 or on cytokine induction at the injection site in either the control or IL-18-treated mice 1 day after infection but that it did modify the host response to HSV-1 infection later, resulting in superficial diminution of the improved survival rate of IL-18-treated mice.
TABLE 5.
Cytokine titers of PWF from vehicle- and IL-18-administered BALB/c mice treated with d- or l-NMMA 26 h after i.p. inoculation with 104 PFU of HSV-1
| In vivo treatment | PWF
|
|
|---|---|---|
| Antiviral activitya (mIFN-α IU/ml) | IFN-γb (101 pg/ml) | |
| d-NMMAc + vehicled | 13 ± 8e | <32 ± 45 |
| l-NMMA + vehicle | 37 ± 57 | <29 ± 33 |
| d-NMMA + IL-18 | <1 ± 0 | <6 ± 0 |
| l-NMMA + IL-18 | <1 ± 0 | <6 ± 0 |
Antiviral activity was measured by using VSV and mouse L929 cells and is expressed as the mIFN-α titer.
Measured by ELISA.
d- or l-NMMA was injected i.p. on days 3, 2, and 1 before infection and days 0 and 1 after infection.
On days 2 and 1 before infection and days 0 and 1 of infection, 1 μg of IL-18 was injected i.p. into each mouse (n = 5 per group), and the mice were sacrificed to prepare PWF 2 h after the final injection with the vehicle or IL-18 and 4 h after the final injection with d- or l-NMMA.
Mean ± standard deviation.
DISCUSSION
Adaptive immunity has been reported to be generally important in immunity to HSV-1 infection (27). The 50% lethal doses of HSV-1 used in the present study were 3.2 log10 PFU for BALB/c mice and 2.4 log10 PFU for BALB/c nude mice, suggesting the importance of T cells for protection in our experimental system also. In the present study, adaptive immunity may have played a protective role because the survival rate of IL-18-treated mice was higher than that of IL-18-treated immunodeficient mice. However, there was no statistically significant difference in survival between BALB/c heterozygous (nu/+) and homozygous nude (nu/nu) mice in HSV-1 infection. Although IL-18 has already been reported to stimulate proliferation and Th1-type cytokine production by activated T cells (18, 28), we found neither enhanced viral clearance nor heightened Th1 cytokine production in IL-18-treated mice more than 3 days after infection. Therefore, we have no evidence to determine whether IL-18 activates adaptive immunity directly or indirectly in vivo or whether the activated adaptive immunity is effective if activated by IL-18. Preliminary experiments revealed that 4 days of treatment with IL-18, which was used in the present study, was more efficient in protection against HSV-1 infection than was posttreatment with IL-18 starting on day 1 or 6 after infection (data not shown). These experiments were conducted to determine whether IL-18 can exert an antiviral effect in the later stage of infection. In the present study, innate immunity was believed to play a key role in IL-18-induced protection against HSV-1 infection since this treatment improved the survival rate of certain mice deficient in adaptive immunity, such as nude and SCID mice, as well as normal infected mice. Collectively, our results suggested that innate immunity rather than adaptive immunity may be important for IL-18-induced protection against HSV-1 infection, at least under the present experimental conditions.
Although IFN-γ production by NK and B cells has been reported in IL-18 treatment in vitro (14, 38), in vivo our treatment induced no appreciable IFN-γ production in BALB/c mice before or after HSV-1 infection. In vivo experiments using IFN-γ −/− mice, however, revealed the involvement of IFN-γ in IL-18-promoted resistance. Furthermore, treatment with IFN-γ in place of IL-18 improved the survival of both BALB/c and BALB/c nude mice with HSV-1 infection (data not shown). In addition to NK and B cells, macrophages from the mouse peritoneal cell pool also produced IFN-γ upon stimulation with IL-18 in vitro (3). Interestingly, IL-18 promoted synergistic IFN-γ synthesis by NK cells, B cells, and macrophages in the presence of IL-12 in vitro (14, 26, 38). In in vitro experiments using mouse peritoneal cells, we found that IL-18 augmented IFN-γ release by naive peritoneal cells more strongly than primed peritoneal cells from mice treated with IL-18. These observations suggest that IFN-γ produced by peritoneal cells stimulated with IL-18 before HSV-1 injection plays an important role in protection in vivo. Studies on the mechanism of regulation of IFN-γ production by peritoneal cells from mice treated with IL-18 are ongoing, and at least two possibilities are being considered: a change in the IFN-γ-producing cell population of peritoneal cells or a change in the sensitivity of peritoneal cells to IL-18. On day 1 of infection, relatively higher titers of antiviral activity and of IFN-γ accompanying the higher HSV-1 titers were found at the injection site in control mice than in IL-18-treated mice. We believe that the higher production of antiviral cytokines such as type I and II IFN may be a host response resulting from the higher HSV-1 replication level in control mice, in contrast to the weak production of such cytokines in response to the low HSV-1 replication level in IL-18-treated mice.
Treatment with anti-asialo GM1 antibody had no influence on either the survival or the inhibition of HSV-1 growth at the injection site in IL-18-treated BALB/c mice or control mice. Since perforin-dependent NK cell activity is known to be effective in viral infections (27, 32), we examined the difference in resistance to HSV-1 infection between C57BL/6 and beige mice, one of whose immune deficiency characteristics is a lack of NK cell activity. Both strains exhibited better resistance to infection with 104 PFU of HSV-1 than did BALB/c mice, but as known previously, beige mice were less resistant than C57BL/6 mice (mortality, 80% versus 40%; n = 10), in which HSV-1 was undetectable in the PWF on day 1 after infection. Selective lysis of virus-infected cells would require the NK cell to bind to the target cell, but regulation of the infection via IFN-γ production may just require the IL-18-activated cells to produce it without contact between the cytokine-activated cells and the virus-modified target cell. Therefore, the above-described observations suggest that IFN-γ production, but not increased NK cell killing, may be the most efficient way to regulate HSV-1 in BALB/c mice treated with IL-18 because HSV-1 replicates better in BALB/c mice than in C57BL/6 mice. Furthermore, IL-18 enhanced IFN-γ production slightly in vitro even by peritoneal cells from mice that underwent anti-asialo GM1 treatment. This finding may explain the resistance to HSV-1 infection in mice treated with both IL-18 and anti-asialo GM1 antibody. Although the modulation of IFN-γ release seen in NRS- or antibody-treated mice 1 day after infection remains to be studied in relation to IL-18-promoted protection, IFN-γ production before infection, even if inhibited to some degree, may be relatively more important for protection against HSV-1 infection than that after infection. The viability of IL-18-treated infected BALB/c mice was 65% (total from four independent experiments, n = 20); thus, there is no significant difference in survival between NRS or antibody-treated mice administered IL-18 and nontreated mice administered IL-18 after HSV-1 infection. Additionally, since some CD8+ T cells and a percentage of peritoneal macrophages have been reported to express asialo GM1 on their surface (21, 29), it was hypothesized that anti-asialo GM1 antibody treatment may damage the functioning of these cells. However, the key role of T-cell-independent immunity, rather than T-cell-dependent immunity, in the protective effect of IL-18 was suggested in our experimental system. A trial of crude carrageenan treatment, but not anti-asialo GM1 antibody treatment, diminished the IL-18-induced resistance to HSV-1 infection (data not shown). Thus, we had no clear evidence supporting the malfunction of T cells and macrophages in anti-asialo GM1 antibody-treated infected mice.
An increase in the serum nitrate concentration and a decrease in virus titers in the organs were reported in ectromelia virus-infected mice (15). In our experiments, the serum nitrate concentration decreased in IL-18-treated mice but increased in control mice on days 1 and 3 of infection. Additionally, peritoneal cells obtained from vehicle-treated mice 1 day after infection produced more NO than those from IL-18-treated infected mice in vitro, whereas nitrate was undetectable in the PWF of both groups. Interestingly, peritoneal cells from vehicle-treated infected IFN-γ −/− mice 1 day after infection produced more NO than those from IL-18-treated infected IFN-γ −/− mice (12.0 versus 1.0 μM; supernatants pooled from triplicate samples), suggesting the presence of IFN-γ-independent NO production. Treatment with l-NMMA had no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. The reason for the noneffectiveness of l-NMMA treatment may be explained at least partly by the poor NO synthesis in IL-18-treated mice. In a mouse model of HSV-1-induced pneumonitis, mice treated with l-NMMA had more virus in the lung than did control mice 3 days after infection, but the lung damage improved 7 days after infection with almost the same virus titers as those in the control mice, revealing not only the protective effect but also the pathogenic effect of NO in vivo (1). Since we also found pneumonitis microscopically in infected mice treated with the vehicle or IL-18 by autopsy, NO may have modified the host response to HSV-1 infection more than 1 day after infection in our experiments also. However, we do not have sufficient evidence to discuss the role of NO in a stage of infection as late as that described above.
The mechanism of IFN-γ-independent interference in the HSV-1 replication observed in IFN-γ −/− mice mice treated with IL-18 on day 1 after infection is unknown. One day of pretreatment with IL-18 (0.001 to 1 μg/ml) did not inhibit HSV-1 replication in J774A.1 macrophages or L929 fibroblasts as well as it did VSV growth in L929 cells compared to the interference with viral replication caused by pretreatment with IFN-γ (data not shown). Furthermore, in the neutralization test using Vero cells, no disturbance of HSV-1 infection was observed when HSV-1 was incubated with IL-18 (0.01 to 10 μg/ml) at 37°C for 1 h, in contrast to the inhibition seen with anti-HSV-1 antibody (data not shown). The possibility of competitive binding by IL-18 and HSV-1 to target cells is also rather unlikely (24, 33). Further research may reveal an IL-18-induced modulation of the functions of other systems known to interact with HSV-1 (30). To understand the mechanism of the antiviral effect of IL-18 completely and to develop a therapeutic application for infectious diseases, further studies should be conducted.
ACKNOWLEDGMENTS
We thank S. Arai, T. Hanaya, and K. Iwaki for helpful discussions, T. Kimoto for valuable pathological information, T. Tatefuji and S. Akamatsu for assistance in statistical analysis, T. Ariyasu for help in preparation of the manuscript, and L. Keleher for editing the manuscript.
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