INTRODUCTION

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Natural killer T cells (NKT cells), a subset of T cells that coexpress NK cell receptors, play important roles in various aspects of immune responses, including regulation of allergic and autoimmune diseases, prevention of tumor metastasis, and protection against bacterial and parasitic infections (3, 7, 12, 15, 18, 37, 42). -Galactosylceramide (-GalCer), a synthetic glycolipid, is recognized in a specific manner by V14⁺ NKT cells (12, 25), which results in the production of both gamma interferon (IFN-) and interleukin-4 (IL-4) (4, 12, 25, 39) and also in the apoptotic death of these cells (9, 34). Recent studies have indicated that ligand-specific activation of V14⁺ NKT cells by -GalCer protects against tumorigenesis and the development of infectious diseases (10, 24, 26, 44).

IL-18 potentiates the production of IFN- by NK and CD4⁺ T cells and acts synergistically with IL-12 in inducing IFN- synthesis by a variety of cells, including NK, T, and B cells and macrophages (8). Recently, IL-18 has been shown to activate IFN- synthesis by NKT cells and cytotoxic activity in collaboration with IL-12 or triggering of T-cell antigen receptor (5, 28). This cytokine by itself does not induce the differentiation of Th1 cells but strongly enhances such responses caused by IL-12 (36). Many investigators indicated that administration of IL-18 rendered hosts resistant to infection by various intracellular microorganisms (20, 22, 23, 30, 33, 45), which is consistent with the notion that IL-18 is involved in the development of Th1 responses. In contrast, Hoshino and coworkers (13) reported that IL-18 acted as a cofactor in inducing the production of IL-13 by NK and T cells caused by IL-2 and that in vivo administration of IL-18 induced immunoglobulin E (IgE) production through the induction of Th2 cytokines (14). Similarly, IL-18 was shown to induce the production of IL-4, IL-13, and histamine by basophils when stimulated with IL-3 and contributed to the production of IgE in mice infected with Nippostrongylus brasiliensis (48).

Cryptococcus neoformans, a ubiquitous fungal pathogen, causes a life-threatening infection of the central nervous system in patients with AIDS. The host resistance to this pathogen is critically regulated by a balance between Th1 and Th2 cytokines; predominant synthesis of Th1 cytokines over Th2 protects mice against infection, whereas infection is exacerbated under Th2 dominant conditions (1, 6, 19, 21, 23). Recently, we demonstrated that administration of -GalCer rendered mice resistant to systemic infection with this pathogen through the induction of IFN- production by innate immune cells and enhancement of fungus-specific Th1 cell development (24). In the present study, we elucidated the role for IL-18 in this response by examining the influence of defective synthesis of this cytokine on Th1 response activated by C. neoformans infection and -GalCer treatment in IL-18-deficient mice. Our results demonstrated that IL-18 defect did not impair the induction of Th1 response but rather resulted in enhanced production of IFN- and IL-12.

	MATERIALS AND METHODS

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Animals. IL-18KO mice were established as described previously (43) and backcrossed eight times to C57BL/6 mice. Breeding pairs of IL-12p40KO mice on a C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, Maine). Mice with a deletion of the genes coding both IL-12p40 and IL-18 were generated by mating IL-12p40KO and IL-18KO mice. These mice were bred in a pathogen-free environment at the Laboratory Animal Center for Biomedical Science, University of the Ryukyus, and all experiments were performed under the same conditions. C57BL/6 mice were purchased from Charles River Japan (Osaka, Japan) and used as a control wild-type (WT) animal. All mice were used at 7 to 13 weeks of age. All experimental protocols described in this study were approved by the Ethics Review Committee for Animal Experimentation of our university.

Microorganisms. A serotype A-encapsulated strain of C. neoformans, designated YC-13, was established from a patient with pulmonary cryptococcosis (47). The yeast cells were cultured on potato dextrose agar plates for 2 to 3 days before use. To induce systemic infection, mice were anesthetized with diethyl ether (Wako Pure Chemical Industries, Osaka, Japan) and injected intravenously with live C. neoformans (10⁶ cells) at 100 µl per mouse.

Culture medium and reagents. RPMI 1640 medium was obtained from Gibco-BRL (Grand Island, N.Y.), fetal calf serum from Cansera (Rexdale, Ontario, Canada). -Galactosylceramide (-GalCer) was kindly provided by Kirin Brewery Co. (Gunma, Japan). The stock solution of -GalCer (220 mg/ml in 0.5% polysorbate 20 in normal saline [NS]) was diluted into 10 µg/ml with NS. Polysorbate 20 solution (0.02% in NS) was used as a control vehicle solution. -GalCer or control solution was injected intraperitoneally at 200 µl per mouse at days 0, 3, and 7 postinfection. Murine recombinant IL-4 was purchased from PeproTech, Inc. (Rocky Hill, N.J.). Neutralizing anti-IL-12 antibody (Ab) (i.e., rabbit IgG) was purchased from R&D Systems (Minneapolis, Minn.). Anti-IL-4 monoclonal Ab (MAb) was purified by using a protein G column kit (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) from culture supernatants of a hybridoma (ATCC clone 11B11). To neutralize endogenously produced IL-12 or IL-4, mice were injected intraperitoneally with each Ab at 400 µg at days 1, 0, and 3 of infection. Rabbit (Wako) or rat IgG (ICN Pharmaceuticals, Inc., Aurora, Ohio) was used as the control Ab, respectively.

In vitro stimulation of spleen cells. Spleen cell suspension was prepared from mice 3 or 7 days after infection with C. neoformans and cultured at 2 × 10⁶/ml with various doses of live microbes for 48 h. The culture supernatants were collected for the measurement of IFN- and IL-4 by enzyme-linked immunosorbent assay (ELISA).

Measurement of cytokines. Murine IFN-, IL-4, IL-12p40, and p70 were measured by using the respective ELISA kit (from Endogen, Inc., Cambridge, Mass., for IFN- and IL-4 and from BioSource International, Inc., Camarillo, Calif., for IL-12p40 and p70). The detection limits of assays for IFN-, IL-4, IL-12p40, and p70 were 15, 5, 2, and 4 pg/ml, respectively.

Enumeration of viable C. neoformans. Mice were sacrificed at day 7 after infection, and spleens and lungs were dissected carefully and excised and then separately homogenized in 10 ml of distilled water by teasing with a stainless mesh. The homogenates, appropriately diluted with distilled water, were inoculated at 100 µl on potato dextrose agar plates and cultured for 2 to 3 days, and the colonies were then counted.

Statistical analysis. Data are expressed as the mean ± the standard deviation (SD). Differences between groups were examined for statistical significance by using the analysis of variance test with a post hoc analysis (Fisher PLSD test). A P value of <0.05 was considered significant.

	RESULTS

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-GalCer induces higher levels of IFN- in the serum of IL-18KO mice than in WT mice. WT and IL-18KO mice were treated with -GalCer or vehicle at days 0, 3, and 7 after intravenous injection of C. neoformans or normal saline, and levels of IFN- in serum were measured at the same time points. As shown in Fig. 1A, in -GalCer-treated WT mice, IFN- levels increased at day 7 and decreased at day 14 postinfection, while in vehicle-treated WT mice, such an increase was not detected at any time points. Unexpectedly, -GalCer treatment induced significantly higher amounts of IFN- at days 3 and 7 postinfection in IL-18KO mice than those in WT mice, but IFN- synthesis decreased to similar levels observed in WT mice at day 14. In uninfected WT mice, -GalCer did not induce any detectable IFN- in the serum at any time points except for day 3, when a marginal level was detected. On the other hand, the same treatment caused the production of higher amounts of IFN- in the sera of IL-18KO mice at day 3 compared with WT mice; these amounts subsequently decreased to basal levels at days 7 and 14 (Fig. 1A). To determine the role of IL-12, we compared the production of IFN- caused by -GalCer in WT and IL-12p40KO mice infected with C. neoformans. As shown in Fig. 1B, a large amount of IFN- was produced in the serum of -GalCer-treated WT mice 7 days after infection, while such production was totally abolished in IL-12p40KO mice.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   -GalCer increases IFN- levels in serum in IL-18KO mice. (A) WT and IL-18KO mice were injected intravenously with C. neoformans (106/mouse) or normal saline. These mice were treated with an intraperitoneal injection of -GalCer (2 µg/mouse) or the same volume of vehicle at days 0, 3, and 7, and the levels of IFN- in serum were measured at the indicated time points. Each symbol represents the mean ± the SD of five mice. Symbols: , vehicle-treated WT mice; , -GalCer-treated WT mice; , vehicle-treated IL-18KO mice; , -GalCer-treated IL-18KO mice. , P < 0.05 compared to -GalCer-treated WT mice. (B) WT and IL-12KO mice were infected with C. neoformans (106/mouse) and then treated with -GalCer intraperitoneally (2 µg/mouse) or a similar volume of vehicle at days 0 and 3. The levels of IFN- in serum were measured 7 days after infection. Each bar represents the mean ± the SD of five mice. Open bars, vehicle treated; closed bars, -GalCer treated.

-GalCer induces enhanced Th1 cell development in IL-18KO mice compared to that in WT mice. To elucidate the role for IL-18 in the fungus-specific Th1 cell development caused by treatment with -GalCer, spleen cells were prepared from WT and IL-18KO mice at days 3 and 7 postinfection, and their IFN- synthesis upon restimulation with microorganisms was measured. As shown in Fig. 2, a small amount of IFN- was induced in spleen cells obtained from -GalCer-treated WT mice at day 3, and such production was markedly enhanced at day 7. In IL-18KO mice, such IFN- synthesis by spleen cells restimulated with 10⁵, 10⁶, and 10⁷ cells of microorganisms was significantly enhanced at day 3, and similar results were obtained in day 7 spleen cells restimulated with 10⁴ and 10⁵ yeast cells. In vehicle-treated conditions, IFN- synthesis by restimulated spleen cells was not significantly different between WT and IL-18KO mice at days 3 and 7 postinfection.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   -GalCer enhances pathogen-specific Th1 response in IL-18KO mice. WT and IL-18KO mice were infected intravenously with C. neoformans (106/mouse) and then treated intraperitoneally with -GalCer (2 µg/mouse) or the same volume of vehicle at days 0 and 3. Spleen cells were prepared at day 3 or 7 and restimulated with the indicated doses of microorganisms for 48 h, and the concentrations of IFN- in the culture supernatants were measured. Each symbol represents the mean ± the SD of three mice. Symbols: , vehicle-treated WT mice; , -GalCer-treated WT mice; , vehicle-treated IL-18KO mice; , -GalCer-treated IL-18KO mice. , P < 0.05 compared to -GalCer-treated WT mice.

-GalCer-enhanced IFN- production is mediated by IL-12 in IL-18KO mice. To elucidate the mechanism of enhanced IFN- production in IL-18KO mice, levels of IL-12p40 in serum were compared at days 0, 3, 7, and 14 between WT and IL-18KO mice. As shown in Fig. 3, vehicle-treated WT mice showed only a marginal change in serum concentrations of IL-12p40 during the course of infection, while -GalCer treatment increased these levels at days 7 and 14. In contrast, IL-18KO mice exhibited significantly higher levels of IL-12p40 in serum by the same treatment at days 3 and 7 postinfection than in WT mice, although no difference was found at day 14. In uninfected mice, no significant difference was observed. The bioactive IL-12p70, however, was not detected in the sera of these mice at any time points postinfection (data not shown). Collectively, these results suggested the possible involvement of IL-12 in such mechanism. In the next step, we tested this conclusion by examining the effect of neutralizing anti-IL-12 Ab on the level of IFN- in serum in IL-18KO mice. As shown in Fig. 4A, this treatment strongly inhibited the increase in IFN- in serum in infected and -GalCer-treated IL-18KO mice, while control rabbit IgG did not show such an effect. In addition, fungus-specific Th1 cell development, as indicated by spleen cell IFN- synthesis, upon restimulation with microorganisms was markedly impaired in infected and -GalCer-treated IL-18KO mice when they were treated with anti-IL-12 Ab but not when treated with control IgG (Fig. 4B). These data indicated that enhanced IFN- production in IL-18KO mice was mediated by increased synthesis of IL-12, although bioactive IL-12 was not detected. This conclusion was further confirmed by using IL-12p40-IL-18 double KO (DKO) mice, which did not synthesize either IL-12 or IL-18. As shown in Fig. 4C, IFN- synthesis caused by -GalCer treatment was significantly higher in IL-18KO mice than in WT mice, and such production was totally abolished in DKO mice.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   -GalCer increases IL-12 in serum in IL-18KO mice. WT or IL-18KO mice were injected intravenously with C. neoformans (106/mouse) or normal saline. These mice were treated with an intraperitoneal injection of -GalCer (2 µg/mouse) or the same volume of vehicle at days 0, 3, and 7, and the levels of IL-12p40 in serum were measured at the indicated time points. Each symbol represents the mean ± the SD of five mice. Symbols: , vehicle-treated WT mice; , -GalCer-treated WT mice; , vehicle-treated IL-18KO mice; , -GalCer-treated IL-18KO mice. , P < 0.05 compared to -GalCer-treated WT mice.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Involvement of IL-12 in -GalCer-induced enhanced production of IFN- in IL-18KO mice. (A) IL-18KO mice were infected intravenously with C. neoformans (106/mouse) and treated with -GalCer (2 µg/mouse intraperitoneally) at days 0 and 3. These mice were treated with 400 µg of anti-IL-12 Ab or the same dose of control IgG at days 1, 0, and 3. The levels of IFN- in serum were measured at day 7 postinfection. (B) In the same experiment, spleen cells were prepared and restimulated with various doses of microorganisms for 48 h, followed by measurement of IFN- concentrations in the culture supernatants. Open bars, medium; hatched bars, 105/ml; striped bars, 106/ml; closed bars, 107/ml. (C) WT, IL-18KO, or DKO mice were infected intravenously with C. neoformans (106/mouse) and treated intraperitoneally with -GalCer (2 µg/mouse) or the same volume of vehicle at days 0 and 3. The levels of IFN- in serum were measured at day 7 postinfection. Each column represents the mean ± the SD of five mice. ND, not detected; NS, not significantly different; , P < 0.05 compared to PBS-treated mice. , P < 0.05 compared to -GalCer-treated WT mice.

Effect of IL-18 deficiency on -GalCer-induced IL-4 production. Because recent investigations indicated the involvement of IL-18 in Th2 response (13, 14, 48), we hypothesized that suppressed production of Th2 cytokines might contribute to the enhanced Th1 response in IL-18KO mice. To test this possibility, we compared IL-4 levels in serum following treatment with -GalCer between WT and IL-18KO mice. As shown in Fig. 5A, the data were not consistent with this hypothesized mechanism, because IL-4 levels were not diminished in the latter group of mice compared to the former under the -GalCer-treated condition but were rather significantly increased at days 7 and 14 in uninfected mice and at day 7 in infected mice. Next, we compared the in vitro synthesis of IL-4 by spleen cells from infected and -GalCer-treated or untreated mice upon restimulation with fungal antigens between WT and IL-18KO mice. Similar to the in vivo experiments, IL-4 production did not diminish but rather was enhanced at 10⁷ yeast cells at day 3 and at 10⁴ and 10⁷ yeast cells at day 7 in IL-18KO mice (Fig. 5B).

View larger version (20K): [in this window] [in a new window]   FIG. 5.   -GalCer increases IL-4 synthesis in IL-18KO mice. (A) WT and IL-18KO mice were injected intravenously with C. neoformans (106/mouse) or normal saline and then treated intraperitoneally with -GalCer (2 µg/mouse) or the same volume of vehicle at days 0, 3, and 7, and the levels of IL-4 in serum were measured at the indicated time points. Each symbol represents the mean ± the SD of five mice. (B) WT and IL-18KO mice were infected intravenously with C. neoformans (106/mouse) and were treated with intraperitoneal injections of -GalCer (2 µg/mouse) or the same volume of vehicle at days 0 and 3. Spleen cells obtained at day 3 or 7 were restimulated with the indicated doses of microorganisms for 48 h, and the concentrations of IL-4 in the culture supernatants were measured. Each symbol represents the mean ± the SD of three mice. Symbols: , vehicle-treated WT mice; , -GalCer-treated WT mice; , vehicle-treated IL-18KO mice; , -GalCer-treated IL-18KO mice. , P < 0.05 compared to -GalCer-treated WT mice.

-GalCer-induced IFN- overproduction is mediated by IL-4 in IL-18KO mice. To elucidate whether IL-4 contributes to the enhanced production of IFN- caused by -GalCer in IL-18KO mice, the infected and -GalCer-treated IL-18KO mice were administered with neutralizing anti-IL-4 MAb and the levels of IFN- in serum were measured at days 3 and 7 after infection. As shown in Fig. 6A, the in vivo synthesis of IFN- in serum was significantly reduced in mice treated with anti-IL-4 MAb, compared with those in control IgG-treated mice, at both time periods. In further experiments, we examined the effect of anti-IL-4 MAb treatment on the in vitro production of IFN-. As shown in Fig. 6B, such treatment significantly attenuated the synthesis of IFN- by spleen cells from infected and -GalCer-treated IL-18KO mice upon restimulation with live microorganisms compared with that by spleen cells from control IgG-treated mice. However, levels of IL-12p40 in serum were not significantly influenced by neutralization of IL-4 (data not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 6.   -GalCer-enhanced production of IFN- in IL-18KO mice is mediated by IL-4. (A) IL-18KO mice were infected intravenously with C. neoformans (106/mouse) and then treated intraperitoneally with -GalCer (2 µg/mouse) at days 0 and 3. These mice were treated with 400 µg of anti-IL-4 MAb or the same dose of control IgG at days 1, 0, and 3. The levels of IFN- in serum were measured at days 3 and 7 postinfection. Each bar represents the mean ± the SD of five mice. (B) In the same experiment, spleen cells were prepared at day 7 and restimulated with various doses of microorganisms for 48 h, and the concentrations of IL-4 in the culture supernatants were measured. Each symbol represents the mean ± the SD of five mice. Symbols: , control IgG-treated; , anti-IL-4 MAb-treated. , P < 0.05 compared to control IgG-treated mice.

View larger version (15K): [in this window] [in a new window]   FIG. 7.   IL-4 increased IFN- synthesis caused by -GalCer in infected mice. (A) WT mice were infected intravenously with C. neoformans (106/mouse) and then treated intraperitoneally with -GalCer (2 µg/mouse) at days 0 and 3. These mice received daily treatment with 2 µg of IL-4 or the same volume of PBS from the day of infection. The levels of IFN- in serum were measured at days 3 and 7. (B) In the same experiment, spleen cells were prepared at day 3 and restimulated with the indicated doses of microorganisms for 48 h, and the concentrations of IFN- in the culture supernatants were measured. Each bar represents the mean ± the SD of three mice. Open bar, PBS-treated; closed bar, IL-4-treated. NS, not significantly different; , P < 0.05 compared to PBS-treated mice.

Effect of IL-18 deficiency on -GalCer-induced host defense against cryptococcal infection. Finally, we examined the effect of IL-18 deficiency on the host defense to cryptococcal infection caused by -GalCer. For this purpose, WT or IL-18KO mice were treated with -GalCer after infection with C. neoformans, and the fungal loads in spleen and lung were measured at day 7 postinfection. In both groups of mice, the numbers of live colonies were significantly decreased in the two organs by this treatment. As shown in Fig. 8, the reduction in spleen loads was significantly more marked in IL-18KO mice than that in control mice, although this difference was not statistically significant in the lung.

View larger version (17K): [in this window] [in a new window]   FIG. 8.   Effect of IL-18 deficiency on -GalCer-induced host defense against cryptococcal infection. WT or IL-18KO mice were infected intravenously with C. neoformans (106/mouse) and then treated intraperitoneally with -GalCer (2 µg/mouse) or the same volume of vehicle at days 0 and 3. At day 7, the numbers of live colonies in the spleen and lung were counted. Results are expressed as the delta fungal loads, which was calculated by subtracting the mean value of log10 CFU in vehicle-treated mice (n = 6; spleen, 4.7 ± 0.2 in WT mice and 4.8 ± 0.1 in IL-18KO mice; lung, 3.4 ± 0.1 in WT mice and 3.6 ± 0.1 in IL-18KO mice) from log10 CFU in -GalCer-treated mice. Each bar represents the mean ± the SD of six mice. Open bars, WT mice; solid bars, IL-18KO mice. NS, not significant; , P < 0.05 compared with WT mice.

	DISCUSSION

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Several studies have focused on the role of NKT cells in tumor immunity, autoimmune diseases, allergic diseases, and infectious immunity, following the discovery of its ligand and its high potential ability to produce both IFN- and IL-4 (4, 12, 16, 25, 39). Several studies revealed the involvement of this lymphocyte subset in host resistance to infectious pathogens (7, 15, 18, 37) and the protective effect of its specific ligand, - GalCer, against Plasmodium yoelii and Plasmodium berghei (10). We also showed previously that activation of V14⁺ NKT cells by -GalCer resulted in the production of IFN- by innate and Th1 cells and development of protective immunity against C. neoformans (24). Recent studies have shown that IL-18 activates IFN- synthesis and cytotoxic activity of NKT cells in collaboration with IL-12 or triggering of T-cell antigen receptor (5, 28), suggesting the possible involvement of this cytokine in the induction of Th1 response by -GalCer. In the present study, therefore, we examined the role for IL-18 in the expression of biological effects of -GalCer. Unexpectedly, we observed enhanced production of IFN- after treatment with this agent in IL-18KO mice, while such production was completely abolished in IL-12KO mice. These results clearly indicated that IL-12, but not IL-18, totally contributed to the induction of Th1 response by -GalCer in mice with experimentally induced systemic cryptococcosis.

In a series of recent studies, we demonstrated that IL-18 plays an important role in the local host defense against infection with C. neoformans in the lungs by enhancing IFN- production by NK cells and potentiating IL-12-induced development of Th1 cells (22, 23). These findings are apparently in contrast to the results of the present study. There are two important differences in the experimental conditions compared to our previous studies. First, we adopted a very potent and specific activator of V14 NKT cells in the present study, which could bypass the requirement of IL-18 for their activation during physiological immune responses in response to natural infection. Another difference is related to the route of infection. For example, Wilder and coworkers (46) showed that intratracheal and intravenous infection resulted in a quite different immune response and host resistance against C. neoformans in C57BL/6 mice. Although the precise mechanism for these inconsistent results on the role of IL-18 remains obscure, some distinction in the immunological milieu between the two routes of infection might produce such opposite outcomes. Further studies are necessary to identify the exact mechanisms underlying these differences.

IL-18 was originally regarded as a cytokine that drives the Th1-Th2 cytokine balance toward Th1 predominant state by inducing IFN- synthesis and potentiating IL-12-induced development of Th1 cells (5, 8, 28, 36). In contrast, in recent studies, this cytokine has been shown to induce immune responses mediated by Th2 cytokines, which antagonize the Th1 responses (32) under particular conditions (13, 14, 48). Furthermore, Leite-de-Moraes et al. (29) have recently indicated the pro-Th2 effect of IL-18 through the induction of IL-4 production by -GalCer-activated NKT cells. They observed that coadministration of IL-18 resulted in increased IL-4 production and activation of B cells caused by -GalCer. Thus, a possible mechanism to explain the enhanced IFN- synthesis in IL-18KO mice could be based on these reported observations, in which the IL-18 defect may result in a shift in cytokine balance toward a Th1 dominant state through a reduction in IL-4 synthesis. However, this is unlikely in the present study because the production of IL-4 was not reduced but rather enhanced in the sera of -GalCer-treated IL-18KO mice. Similar results were obtained in spleen cells from infected and -GalCer-treated mice. In our study, high IL-12 levels in serum were noted in IL-18KO mice relative to those in WT mice. As demonstrated by Yoshimoto et al. (48), IL-18 could not act as the inducer of Th2 response under conditions in which IL-12 is fully produced.

Interestingly, administration of neutralizing anti-IL-4 MAb inhibited the enhanced production of IFN- in serum and by spleen cells of infected and -GalCer-treated IL-18KO mice. Such effects were not associated with an apparent increase of IL-12p40 synthesis in serum. In contrast, neutralization of IL-4 did not show any significant influence on IFN- synthesis in WT mice (data not shown). Conversely, treatment with IL-4 increased the synthesis of IFN- in WT mice with cryptococcosis and treated with -GalCer. These observations were inconsistent with the previous findings that IL-4 attenuated the production of IL-12 by macrophages and dendritic cells (DCs) (27, 40, 41). However, in recent studies, IL-4 was shown to potentiate the production of IL-12p70 by DCs, although the opposite effect was observed in IL-12p40 (11, 17). In addition, based on the possible role of IL-4 as an inducer of IL-12p70 production, IL-4KO mice showed impaired host resistance to Candida albicans infection associated with attenuated production of IFN- and IL-12 (31). Similar findings were recently reported by Schuler et al. (38), who demonstrated that Th1-mediated tumor immunity was impaired in IL-4KO mice and that administration of exogenous IL-4 resulted in the recovery of the reduced host defense to tumor cells. These observations suggest that endogenously synthesized IL-4 may contribute to the enhanced production of bioactive IL-12p70 by DCs, leading to the development of Th1 response in IL-18KO mice. To confirm this possibility, further studies will be necessary, particularly since the production of IL-12p70 could not be detected in the present study.

Alternatively, quantitative or qualitative difference in NKT cells and DCs should be considered. The presence of large numbers of these cells in IL-18KO mice should enhance Th1 responses. To address this possibility, we compared the proportion of NKT cells and DCs in spleen and liver between the two strains of mice. Our results indicated that there were no significant differences in such values in the spleen (NKT cells [1.2% ± 0.2% and 1.2% ± 0.1%] and DCs [0.8% ± 0.1% and 1.0 ± 0.3%] in WT and IL-18KO mice, respectively [n = 3]) and liver (NKT cells [25.4 ± 1.19% and 24.0 ± 0.8%] and DCs [0.4 ± 0.5% and 0.5 ± 0.1%] in WT and IL-18KO mice, respectively [n = 3]). Furthermore, we evaluated the ability of DCs to produce IL-12 upon stimulation with various microbial products, including mannoproteins and galactoxylomannan, major immunostimulating components of C. neoformans (2), as well as lipopolysaccharide in WT and IL-18KO mice, because IFN- production caused by -GalCer in the present study was totally dependent on this cytokine. In a recent studies, Pitzurra et al. (35) showed that these cryptococcal products activated human peripheral blood monocytes to produce IL-12. Thus, the more profound synthesis of IL-12 by DCs by cryptococcal products could result in enhanced induction of Th1 response. However, we did not detect any difference in the production of both IL-12p40 and p70 by DCs derived from WT and IL-18KO mice (data not shown). Furthermore, there was no significant difference in IFN- synthesis by purified hepatic NKT cells from WT or IL-18KO mice when cultured with -GalCer-pulsed DCs from either strain of mice (data not shown). Based on these findings, it was difficult to explain the -GalCer-induced increase in Th1 response in IL-18KO mice based on proportional and functional differences between the NKT cells and DCs of WT mice. It is possible that IL-18KO mice may be more sensitive to IL-12 than WT mice. To approach this, we examined IFN- synthesis by spleen cells stimulated by various concentrations of IL-12 in the presence or absence of concanavalin A. However, this was not the case because no significant difference was found in the sensitivity to IL-12 between these mice (data not shown).

The recent study of Kawakami et al. demonstrated the protective effect of -GalCer against systemic infection with C. neoformans through the induction of IFN- production (24). This observation suggested that IL-18KO mice may acquire a higher resistance to this infection by -GalCer treatment than WT mice. The results were compatible with such a hypothesis, i.e., that elimination of microorganisms from the spleen was more marked in the former group of mice than in the latter group. Thus, lack of IL-18 synthesis resulted not only in increased production of IFN- but also in enhanced host resistance under particular conditions in which NKT cells are strongly activated.

In conclusion, the present study demonstrates that in the absence of IL-18 synthesis, Th1 response was not reduced, but rather enhanced, through increased production of IL-12 after intravenous infection with C. neoformans and a ligand-specific activation of NKT cells. This observation is likely due to increased synthesis of IL-4, although the precise mechanism remains unclear. Our data suggested the limited contribution of IL-18 in the development of Th1 response and host defense after NKT cell activation during systemic cryptococcosis. However, to understand the precise role of this cytokine in the regulation of NKT cell-mediated host defense, further studies conducted under physiological conditions are necessary.