Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, E-28049 Madrid, Spain
Correspondence
Mariano Esteban
mesteban{at}cnb.uam.es
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ABSTRACT |
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Present address: Laboratorio Reumatologia, Unidad de Investigacion, Hospital 12 de Octubre, Madrid, Spain.
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INTRODUCTION |
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Several studies have analysed the role of different cytokines during infection with vaccinia virus (VV): some increased virulence, as with IL-4, whereas others attenuated the virus, as with IFN- or TNF-
(Ramshaw et al., 1997
). We have described previously that in mice inoculated with recombinant VV (rVV) expressing IL-12, this cytokine attenuates the virus by inducing high levels of IFN-
and incrementing both the antiviral CTL response as well as the specific Th1 : Th2 ratio (Gherardi et al., 1999
). Moreover, another report (van den Broek et al., 2000
) also pointed out the importance of this cytokine in VV infections and suggested that IL-12 is more important than IFN-
against VV. IL-18 displays similar effects as IL-12; thus, it is tempting to speculate that IL-18 might have a protective role against VV infection. In this sense, it has been described that administration of recombinant IL-18 to mice infected with VV induces an antiviral effect mediated by different mechanisms that include NK and CTL cells (Tanaka-Kataoka et al., 1999
). Interestingly, the potential importance of this cytokine against poxvirus infections is suggested by the finding of different poxvirus genes related to IL-18 that contribute to virus evasion of the host immune system (Xiang & Moss, 1999
; Bowie et al., 2000
; Symons et al., 2002
).
While synergy between IL-12 and IL-18 has been observed in different model systems (Fukao et al., 2000; Yamanaka et al., 1999
; Hofstra et al., 1998
), there is little information of this effect in the context of a virus infection. The aim of this study was to analyse the effect of IL-18, either alone or in combination with IL-12, on VV infection in the murine model. We have defined the importance of both cytokines delivered from rVV in virus clearance and in the generation of non-specific and specific anti-VV immune responses.
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METHODS |
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Immunizations of mice and serum sample collection.
BALB/c mice (H-2d), SCID BALB/c mice or C57BL/6 (H-2b) mice, 6 to 8 weeks old, were inoculated intraperitoneally with different doses of the different rVVs in 200 µl sterile PBS. Blood was obtained at different times post-infection (p.i.) from the retroorbital plexus. Serum was then isolated and stored at -20 °C.
Measurement of luciferase activity in mouse tissues.
Replication of rVVs in different mouse tissues was followed by a highly sensitive luciferase assay that correlates with virus titres in vivo (Rodríguez et al., 1988). Briefly, organs from euthanized animals were homogenized in luciferase extraction buffer (300 µl per spleen and 100 µl per ovary) (Promega) and cleared supernatants were obtained. Luciferase activity was measured according to manufacturer's instructions in a Lumat LB 9501 Berthold luminometer (Berthold) and was expressed as relative luciferase units (RLU) mg-1 protein. Protein content in tissue extracts was measured with the Bicinchoninic Acid Protein Assay kit (Pierce).
Evaluation of cytokine levels by ELISA.
Cytokine levels were evaluated in serum and in clarified spleen homogenates, performed in PBS containing protease inhibitors (Complete, Roche). ELISA determinations used the appropriate combination of antibodies and followed the instructions of the manufacturer (Pharmingen).
Evaluation of CD8+ T cells by the ELISPOT assay.
The ELISPOT assay to detect antigen-specific CD8+ T cells was performed as described previously (Ramírez et al., 2000). Antibodies used were anti-mouse IFN-
mAb R4-6A2 and biotinylated anti-mouse IFN-
mAb XMG1.2 (Pharmingen). P815 cells, a mastocytome cell line that expresses only MHC class I molecules (Miyahira et al., 1995
) were used as APCs. The number of specific CD8+ T cell anti-VV antigens was evaluated by infecting P815 cells at an m.o.i. of 5 p.f.u. per cell. At 4·5 h p.i., cells were washed and treated with mitomycin C (30 µg ml-1) (Sigma). Development was performed with peroxidase-labelled avidin (Sigma) by adding 3,3'-diaminobenzidine tetrahydrochloride (Sigma). Spots were counted with the aid of a stereomicroscope.
Antibody measurements by ELISA.
ELISA was used to determine the presence of antibodies against VV antigens in serum samples. VV antigens (1 µg ml-1) used to coat 96-well flat-bottomed plates consisted of envelope proteins extracted from purified virions, as described previously (Rodríguez et al., 1997). Peroxidase-conjugated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology) antibodies were diluted 1 : 1500 and 1 : 2000, respectively, in blocking buffer and incubated for 1 h at 37 °C. Finally, absorbance values were measured at 492 nm on a Labsystems Multiskan Plus plate reader.
NK cell depletion.
NK cells were depleted from 6- to 8-week-old C57BL/6 mice 1 day before virus inoculation. Mice were inoculated intraperitoneally with 100 µg purified mAb PK136 in PBS (1 mg ml-1), which is specific for the pan-NK surface marker NK-1.1 (a generous gift from Werner Held, Ludwig Institute, Lausanne, Switzerland), or with normal serum. NK cell depletion was measured by FACS analysis using the pan-NK DX5 antibody (Pharmingen) in splenocyte suspensions. Levels of depletion were consistently between 60 and 80 % (data not shown).
Statistical analyses.
Statistical analyses were performed by applying the unpaired Student t-test. Significant differences were defined with P<0·01 and P<0·05.
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RESULTS |
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IL-12 and IL-18 delivered from rVV promote a synergistic attenuation of the virus
Next, we analysed if simultaneous expression of IL-12 and IL-18 from different rVVs could enhance the antiviral efficacy of a single cytokine. Four groups of BALB/c mice were inoculated intraperitoneally with different combinations of rVVs; all animals received a total dose of 6x107 p.f.u. of virus expressing the luciferase reporter gene (Fig. 3). The dose of 1x107 p.f.u. rVVIL-12 was chosen to diminish IL-12-mediated anti-VV effects observed previously (Gherardi et al., 1999
) and, thus, allowed us to monitor its potential benefit during the expression of both cytokines. At different days p.i., levels of luciferase activity were measured in the ovaries and spleen (Fig. 3
). In ovaries from mice co-inoculated with the rVVs expressing IL-12 and IL-18 (group IV), there is, from days 2 to 7 p.i., a decrease in virus replication compared to the groups inoculated with rVVs expressing either IL-12 (group III) or IL-18 (group II). Higher differences between groups were observed at 3 and 4 days p.i. Luciferase activities in groups III and IV were significantly different relative to control-infected mice (P<0·01) (rVVHA-) from 2 days p.i. up to 7 days p.i., whereas rVVIL-18-infected mice showed a 6- and 9-fold drop (P<0·01) in luciferase activity at 4 and 7 days p.i., respectively, compared with rVVHA--infected mice. We observed in ovaries at both 3 and 4 days p.i. that co-delivery of rVVIL-12 and rVVIL-18 induced a synergistic activity. Thus, compared to group I (control group) and at 3 days p.i., mice from group IV (rVVIL-12+rVVIL-18) showed 300-fold lower levels of luciferase, while mice inoculated with either rVVIL-18 or rVVIL-12 had 3 and 15 times less luciferase activity, respectively. At this time-point, differences between groups IV and III were significantly different (P<0·05). Moreover, at 4 days p.i., luciferase activity in group IV was nearly 250-fold lower than that in the control group, whereas in groups III and II, the drop was 50- and 6-fold, respectively. When luciferase activity was measured in the spleen, greater differences between the groups were observed at 2 days p.i. This is probably due to the faster kinetics of VV clearance in this organ than in ovaries. In the spleen, no significant differences in luciferase levels were found between animals given rVVIL-18 (group II) with respect to those given the control, rVVHA- (group I). However, data between groups III and IV with respect to group I were statistically significant (P<0·01) at 2 and 3 days p.i. At 2 days p.i., luciferase expression in the spleen of animals from group IV was 50-fold lower than that in control mice, whereas in mice from group III these values were 5-fold lower (10-fold lower levels of luciferase in group IV than in group III, P<0·05). Thus, these data showed that at three time-points p.i., namely days 3 and 4 in the ovaries and day 2 in the spleen, co-delivery of IL-12 and IL-18 induced a much stronger antiviral effect (nearly 10 times higher) than what would be expected from the additive action of both cytokines.
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Pattern of cytokines induced after expression of IL-18 and IL-12 from rVVs
Since the delivery of a cytokine from rVV can affect the expression of other cytokines, we next analysed the levels of Th1 cytokines (IL-12, IL-18 and IFN-) induced in serum and spleen. Mice were inoculated with rVVs expressing IL-12 and IL-18, as described in Fig. 3
, and the cytokine levels in serum and spleen were measured at various times p.i. As observed in Fig. 4
, at shorter times p.i. (6 h), IFN-
levels in the serum were elevated but were similar between groups III and IV. At later times p.i. (16 and 24 h), mice receiving both viruses showed the highest difference in levels of IFN-
(nearly a 2-fold increase) with respect to the group inoculated with rVVIL-12. Thereafter, no appreciable differences were observed between the groups. The production of high levels of IFN-
by co-expression of IL-12 and IL-18 was also supported by the findings obtained in spleen homogenates at 1 day p.i.; however, at 2 and 3 days p.i., the differences between the groups were less evident (Fig. 4
).
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The combined action of IL-12 and IL-18 delivered from rVVs results in enhanced specific humoral and cellular immune responses to VV
Our next aim was to study the consequences that co-expression of IL-12 and IL-18 from rVVs might have on induction of specific anti-VV humoral and cellular immune responses. Mice were inoculated intraperitoneally with rVVs expressing IL-12 or IL-18, following the scheme depicted in Fig. 5. At 10 days p.i., the CD8+ T cell immune response against VV was evaluated by a modified ELISPOT assay that quantifies IFN-
-secreting CD8+ T cells (Fig. 5A
) (Gherardi et al., 1999
) and, at 14 days p.i., the specific anti-VV IgGs in the sera were determined by ELISA (Fig. 5B
). As shown in Fig. 5(A)
, mice inoculated with both rVVIL-12 and rVVIL-18 (group IV) triggered an enhanced specific CD8+ T cell response against VV when compared to mice inoculated with rVVIL-18 (group II) (P<0·01) or rVVIL-12 (group III) (P<0·01) alone. The increment in IFN-
-secreting cells induced independently by each cytokine was 1·6 (rVVIL-18) or 2·7 (rVVIL-12) times higher than in the control group. Thus, the 4-fold increase observed in the specific CD8+ T cell response of group IV is in line with an additive effect of both cytokines. When the levels of specific IgG subclasses induced in the different groups were evaluated 14 days after rVV inoculation, group IV gave the highest ratio (IgG2a : IgG1) of specific IgGs (Fig. 5B
). The increment observed in this group was significantly different (P<0·05) with respect to the response observed in the other three groups. Based on the findings of Fig. 5
, we conclude that the concerted action of both IL-12 and IL-18 delivery in combination by rVVs is more effective in promoting a specific Th1 type of immune response to VV than when each cytokine was delivered individually by the recombinant virus.
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DISCUSSION |
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Interestingly, some authors (van den Broek et al., 2000) have found that high and low susceptibility to VV infection correlate with the absence and presence of CTL responses in IL-12-/- and IFN-
-/- mice, respectively. We have found that co-delivery of both cytokines by rVVs induces an enhanced specific antiviral CD8+ T cell response when compared to mice given rVVIL-12 or rVVIL-18 alone. We also found that rVVIL-12+rVVIL-18 treatment favours the increment of the specific Th1 : Th2 (IgG2a : IgG1) ratio. The importance of T cells on IL-12+IL-18-mediated VV clearance was demonstrated further by experiments with SCID mice. Previous studies have demonstrated that IL-18 plays an important role in the generation of type I effector CD8+ T cells in a CD4+ T cell-dependent manner (Okamoto et al., 1999
) and suggested that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12. Both regulatory mechanisms might be operating in mice inoculated with rVVs co-expressing both cytokines. Moreover, independent and synergistic effects of IL-18 and IL-12 in augmenting cytotoxic T lymphocyte responses and IFN-
production in ageing have also been demonstrated (Zhang et al., 2001
).
NK cells play a critical role in innate immunity against pathogens, virus-infected cells and tumours through MHC-unrestricted cytotoxicity and production of cytokines. Positive synergy has been demonstrated between IL-12 and IL-18 in NK proliferation, cytotoxicity and IFN- production (Tomura et al., 1998
; Lauwerys et al., 1999
) and in mice lacking both IL-12 and IL-18, NK activity and Th1 responses were impaired further (Takeda et al., 1998
). Our findings indicate that NK cell depletion affected the IL-12/IL-18 synergism in the anti-VV protective effect in ovaries, suggesting an important role for NK cells in such synergism. Indeed, this unique effect on NK cells relies on the combined action of both cytokines, as rVV replication is not affected after NK depletion in mice given control rVV, rVVIL-12 or rVVIL-18 alone. To this end, it has been described recently that NK cells derived in the presence of IL-12 and IL-18 displayed strong and unique cytotoxicity, involving the induction of apoptosis and higher expression of perforin compared to NK cells derived in the presence of IL-12 or IL-15 (Lauwerys et al., 2000
). Our data, and those reported by others (Mahalingam et al., 1999
), show that elimination of NK cells had a negligible effect on the control of VV. However, the elimination of NK cells exacerbates the infection with chemokine-encoding rVV, suggesting that NK depletion only affects rVV replication when cytokines or chemokines influencing the antiviral effect of NK functions are expressed from rVVs.
In conclusion, we have described in the mouse model that when IL-12 and IL-18 are co-delivered from rVVs, a synergism in the protective antiviral effect occurs. T and NK cells are involved in such an effect: SCID mice showed a less pronounced clearance of the recombinant virus compared to normal BALB/c mice and NK depletion abrogated the synergism in clearance of the virus. Importantly, besides virus clearance, a bias of the specific anti-VV immune response towards a Th1 type was obtained, as mice inoculated with rVVIL-12+rVVIL-18 showed an enhanced specific CD8+ T cell response as well as an incremented IgG2a : IgG1 ratio. These findings are of interest for the design of both therapeutic and prophylactic vaccines based on poxvirus vectors.
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ACKNOWLEDGEMENTS |
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Received 23 January 2003;
accepted 2 April 2003.