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Infection and Immunity, January 2001, p. 213-220, Vol. 69, No. 1
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.1.213-220.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Activation of Valpha 14+ Natural Killer T Cells by alpha -Galactosylceramide Results in Development of Th1 Response and Local Host Resistance in Mice Infected with Cryptococcus neoformans

Kazuyoshi Kawakami,1,* Yuki Kinjo,1 Satomi Yara,1 Yoshinobu Koguchi,1 Kaori Uezu,1 Toshinori Nakayama,2 Masaru Taniguchi,2 and Atsushi Saito1

The First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa,1 and CREST (Core Research for Evolutional Science and Technology) Project, Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba,2 Japan

Received 15 May 2000/Returned for modification 30 June 2000/Accepted 5 October 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We examined the effect of alpha -galactosylceramide (alpha -GalCer) on the synthesis of gamma interferon (IFN-gamma ) and local resistance in mice infected intravenously with Cryptococcus neoformans. The level of IFN-gamma in serum increased on day 3, reached a peak level on day 7, and decreased to the basal level on day 14 postinfection in mice treated with alpha -GalCer, while in vehicle-treated mice, no increase was detected at any time points except for a small increase on day 7. Such effects were not observed in NKT-KO mice. In CD4KO mice, minor synthesis of IFN-gamma was detected on day 3 in sera but was completely abolished by day 7. The alpha -GalCer-induced IFN-gamma production on day 3 was partially reduced in mice depleted of NK cells by treatment with anti-asialo-GM1 antibody (Ab). Spleen cells obtained from infected and alpha -GalCer-treated mice on day 7 produced a large amount of IFN-gamma upon restimulation with live organisms, while only a marginal level of production was detected in splenocytes from infected and vehicle-treated mice. Such effects were abolished in CD4KO and NKT-KO mice. Finally, the fungal loads in the lungs and spleen on days 7 and 14 were significantly reduced in alpha -GalCer-treated mice compared to those in control mice. In NKT-KO mice, local resistance elicited by alpha -GalCer was completely abolished, although no obvious exacerbation of infection was detected. Furthermore, treatment with anti-IFN-gamma monoclonal Ab mostly abrogated the protective effect of this agent. Thus, our results indicated that activation of Valpha 14+ NKT cells resulted in an increased Th1 response and local resistance to C. neoformans through production of IFN-gamma .


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Natural killer T (NKT) cells are a unique population of lymphocytes distinct from T, B, and NK cells in mice and are recognized by coexpression of both alpha beta T-cell and NK-cell receptors. These cells are typically characterized by the expression of a single invariant alpha  chain of antigen receptor encoded by a rearranged Valpha 14-Jalpha 281 gene segment coupled with a highly skewed beta  chain, such as Vbeta 8.2, Vbeta 7, or Vbeta 2 (2, 13, 17, 27-29). NKT cells play an important role in various aspects of the regulation and effector arms of the immune response, including the regulation of allergic and autoimmune diseases (8, 16, 30, 38), prevention of tumor metastasis (9, 24, 35, 36), and protection against bacterial and parasitic infections (12, 14, 19, 31). In addition, NKT cells are thought to be involved in granuloma formation caused by deproteinized cell wall components of Mycobacterium tuberculosis (1) and in liver injury induced by Salmonella infection (18).

alpha -Galactosylceramide (alpha -GalCer), a synthetic glycolipid originally isolated from marine sponges, is recognized in a specific manner by Valpha 14+ NKT cells, which results in the production of both gamma interferon (IFN-gamma ) and interleukin-4 (IL-4) (10, 23, 32). In early studies by Yoshimoto et al. (40), NKT cells were thought to be the major IL-4-producing cells for the early induction of Th2-cell development from antigen-specific naive T cells. However, more recent studies have shown that CD1- or beta 2-microglobulin-deficient mice, which have markedly reduced numbers of NKT cells, generate comparable levels of antigen-specific Th2 response as a control (4, 7, 10, 15, 33, 42). In addition, antigen-specific induction of the Th2 response was not impaired in Jalpha 281-deficient mice lacking Valpha 14+ NKT cells, and activation of these cells by alpha -GalCer inhibited such a response through the production of IFN-gamma (10).

Cryptococcus neoformans, a ubiquitous fungal pathogen, causes a life-threatening infection of the central nervous system in patients with impaired cell-mediated immunity, such as AIDS (34). 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 the infection is exacerbated under Th2-dominant conditions (3, 6, 11, 20, 21, 41). In recent studies (11, 22), targeted disruption of the gene for IL-12 or IL-18, both of which play important roles in the differentiation of Th1 cells and IFN-gamma synthesis by T and NK cells, resulted in reduced host resistance and Th1 response to C. neoformans. However, the role of NKT cells in the development of fungus-specific Th1 cells and IFN-gamma synthesis during the infection remains to be elucidated.

In the present study, we examined the effects of alpha -GalCer on serum levels of IFN-gamma during systemic infection with C. neoformans in wild-type (WT) and Jalpha 281-deficient (NKT-KO) mice. Furthermore, the cellular source of IFN-gamma production by alpha -GalCer was determined at the early and late phases of infection using NK cell-depleted and CD4KO mice. We also elucidated the mechanisms by which alpha -GalCer treatment influenced the induction of C. neoformans-specific Th1-cell development during infection. Finally, we examined the effects of this treatment on the local resistance to this pathogen by counting the number of live microorganisms in the lungs and spleen.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Animals. Breeding pairs of CD4-deficient (CD4KO) mice on a C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, Maine). Valpha 14+ NKT-cell-deficient (NKT-KO) mice were established by targeted deletion of the Jalpha 281 gene segment (24) and backcrossed eight times with C57BL/6 mice. These mice were bred in a pathogen-free environment in the Laboratory Animal Center for Biomedical Science, University of the Ryukyus. C57BL/6 mice were purchased from SLC Japan (Hamamatsu, Japan) and used as a control WT animal. All mice were used at 7 to 13 weeks of age. All experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of our university.

Microorganisms. A serotype A-encapsulated strain of C. neoformans, designated as YC-13, was established from a patient with pulmonary cryptococcosis (39). Infection with this pathogen was self-limited in the lungs of WT mice and did not disseminate to the brain. 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 was obtained from Cansera (Rexdale, Ontario, Canada), concanavalin A (ConA) and purified protein derivative (PPD) were purchased from Sigma Chemical Co. (St. Louis, Mo.) and Japan BCG Co. (Tokyo, Japan), respectively. alpha -GalCer was provided by Kirin Brewery Co. (Gunma, Japan) and prepared as described previously (25, 26). The stock solution of alpha -GalCer (220 µg/ml in 0.5% polysorbate 20 in normal saline [NS]) was diluted to 10 µg/ml with NS. Polysorbate 20 solution (0.02% in NS) was used as a control vehicle solution. alpha -GalCer or control solution was injected intraperitoneally at 200 µl per mouse on days 0, 3, and 7 postinfection.

Antibodies. Anti-asialo-GM1 (ASGM1) polyclonal antibody (Ab) was purchased from Wako Pure Chemical Industries. To deplete NK cells, mice were injected intraperitoneally with anti-ASGM1 Ab at 200 µg on days -3, 0 and 3 postinfection. Rabbit immunoglobulin G (IgG) (Wako Pure Chemical Industries) was used as the control Ab. In a series of preliminary experiments, we confirmed that such treatment almost completely depleted NK cells, but not NKT cells, as previously reported by Tsukahara et al. (37). Thus, NK (CD3-NK1.1+) cells were reduced from 2.8% ± 0.3% to 0.3% ± 0.1% in the spleen and from 7.6% ± 1.5% to 0.2% ± 0.1% in the liver (n = 3 each), while the proportion of NKT (CD3+ NK1.1+) cells did not change in either organ (1.3% ± 0.1% versus 1.2% ± 0.2% and 15.2% ± 3.1% versus 14.7% ± 1.8% [n = 3 each], respectively). Anti-IFN-gamma monoclonal antibody (MAb) was purified with a protein G column kit (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) from culture supernatants of a hybridoma (clone R4-6A2, purchased from American Type Culture Collection). To block endogenously synthesized IFN-gamma , mice were injected intraperitoneally with 200 µg of this MAb on days -1, 0 and 3 postinfection. Rat IgG (ICN Pharmaceuticals, Inc., Auora, Ohio) was used as a control Ab.

In vitro stimulation of spleen cells. Spleen cells were prepared from mice on day 3 or 7 after infection with C. neoformans and cultured at 2 × 106/ml with various doses of live microorganisms or 1 µg of ConA per ml for 48 h. The culture supernatants were collected, and the concentration of IFN-gamma was measured in these samples by using enzyme-linked immunosorbent assay (ELISA) (Endogen, Inc., Cambridge, Mass.). The sensitivity of the assay was 15 pg/ml. In some experiments, spleen cells were depleted of NK and NKT cells by magnetic separation using sheep-anti-mouse IgG Ab-coated Dynabeads M-450 (Dynal A.S., Oslo, Norway) preincubated with anti-NK1.1 MAb (mouse IgG: purified with a protein G column kit from culture supernatants of a hybridoma [clone PK136, purchased from American Type Culture Collection]). These procedures were followed by the steps recommended by the manufacturer. Control cells were treated with irrelevant mouse IgG (ICN Pharmaceuticals, Inc.) instead of anti-NK1.1 MAb. Flow cytometric analysis revealed that the proportions of NK and NKT cells were reduced from 3.0% ± 0.6% to 0.3% ± 0.1% and from 1.0% ± 0.2% to 0.2% ± 0.1% (n = 3 each), respectively, by depletion with anti-NK1.1 MAb, while control IgG did not change the proportions of these cells (NK cells, 3.0% ± 0.6% versus 2.6% ± 0.1%; NK1.1 cells, 1.0% ± 0.2% versus 0.9% ± 0.1% [n = 3 each] in untreated and control IgG-treated spleen cells, respectively).

Enumeration of viable C. neoformans. Mice were sacrificed on day 7 or 14 after infection, and the lungs and spleens were dissected out carefully, excised, and 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 counted.

Statistical analysis. Data were analyzed using Statview II software (Abacus Concept, Inc., Berkeley, Calif.) on a Macintosh computer. Data are expressed as mean ± standard deviation (SD). Statistical analysis between groups was performed using the analysis of variance test with a post hoc analysis (Fisher PLSD test). A P value less than 0.05 was considered significant.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Induction of IFN-gamma synthesis in sera of mice treated with alpha -GalCer. To elucidate the effect of alpha -GalCer treatment on IFN-gamma synthesis during infection, mice were treated with this agent or control vehicle on days 0, 3, and 7 after intravenous injection of NS or C. neoformans. Levels of IFN-gamma in serum were measured on days 0, 3, 7, and 14. As shown in Fig. 1A, IFN-gamma was not detected before infection, but alpha -GalCer treatment clearly induced its production on day 3, and then it reached a peak level on day 7 and decreased close to the basal level by day 14 in mice infected with C. neoformans. In contrast, only a marginal level of IFN-gamma was detected on day 7 in infected and vehicle-treated mice. On the other hand, the levels of IFN-gamma on day 3 in the sera of alpha -GalCer-treated and uninfected mice were similar to those in the sera of infected and treated mice, and such production rapidly decreased below the detection limit on days 7 and 14 in the former group of mice (Fig. 1B).


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  		FIG. 1.   alpha -GalCer treatment increases the IFN-gamma level in serum. Mice received intraperitoneal injections of alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0, 3, and 7 after intravenous injection of C. neoformans (106/mouse) (A) or the same volume of normal saline (B). On days 0, 3, 7, and 14, the mice were sacrificed and the levels of IFN-gamma in serum were measured. Each symbol represents the mean and SD for three mice. Experiments were repeated three times with similar results. Open circles, vehicle; solid circles, alpha -GalCer. *, P < 0.05 compared with vehicle-treated mice.

To examine the mechanism of alpha -GalCer-induced IFN-gamma synthesis, WT and NKT-KO mice were treated with vehicle or this agent on days 0 and 3 after infection with C. neoformans, and the levels of IFN-gamma in serum were measured on day 7. As shown in Fig. 2, alpha -GalCer treatment induced a marked increase in the production of IFN-gamma in WT mice but not in vehicle-treated mice. In contrast, alpha -GalCer failed to change serum IFN-gamma levels in NKT-KO mice.


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  		FIG. 2.   alpha -GalCer treatment causes NKT-cell-dependent induction of IFN-gamma . WT and NKT-KO mice received intraperitoneal injections of alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0 and 3 after intravenous injection of C. neoformans (106/mouse). On day 7, the mice were sacrificed and the levels of IFN-gamma in serum were measured. Each bar represents the mean and SD for three mice. Experiments were repeated twice with similar results. NS, not significant; *, P < 0.05 compared with vehicle-treated mice.

Cellular source of alpha -GalCer-induced IFN-gamma production. In the next experiment, we determined the cellular source of IFN-gamma synthesis induced by alpha -GalCer. For this purpose, WT and CD4KO mice were treated with the vehicle only or alpha -GalCer at a similar time schedule after infection with C. neoformans, and levels of IFN-gamma in serum were measured on days 3 and 7. In WT mice, a considerable amount of IFN-gamma was detected in the sera of alpha -GalCer-treated mice on day 3 postinfection and the level further increased on day 7, while no IFN-gamma production was detected at both time points in vehicle-treated mice. IFN-gamma production was higher in CD4KO mice than in WT mice on day 3 after infection. Interestingly, however, almost no IFN-gamma production was noted on day 7 in the former mice (Fig. 3). To further examine the cellular source of IFN-gamma production, WT mice were deleted for NK cells by treatment with anti-ASGM1 Ab. As shown in Fig. 4A, NK cell depletion significantly inhibited IFN-gamma production on day 3 but had no influence on day 7 after infection (Fig. 4B).


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  		FIG. 3.   Effect of alpha -GalCer treatment on the IFN-gamma in serum in CD4KO mice. WT and CD4KO mice received intraperitoneal injections of alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0 and 3 after intravenous injection of C. neoformans (106/mouse). On days 3 and 7, the mice were sacrificed and the levels of IFN-gamma in serum were measured. Each bar represents the mean and SD for three mice. Experiments were repeated four times with similar results. ND, not detected.


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  		FIG. 4.   Effect of NK-cell depletion on a alpha -GalCer-induced IFN-gamma production. WT mice received intraperitoneal injections of alpha -GalCer (2 µg/mouse) on days 0 and 3 after intravenous injection of C. neoformans (106/mouse). These mice were injected intraperitoneally with phosphate-buffered saline (PBS), rabbit IgG, or anti-ASGM1 Ab on days -3, 0, and 3 after infection. On days 3 (A) and 7 (B), the mice were sacrificed and the levels of IFN-gamma in serum were measured. Each bar represents the mean and SD for three mice. Experiments were repeated twice with similar results. ND, not detected. NS, not significant; *, P < 0.05 compared with PBS-treated mice.

Induction of Th1 cells specific for C. neoformans by alpha -GalCer. To elucidate whether Th1 cells specific for C. neoformans were induced by treatment with alpha -GalCer, spleen cells were prepared from WT and CD4KO mice treated with vehicle or this agent on day 3 or 7 after infection and assayed for IFN-gamma production upon restimulation with live microorganisms. No detectable amount of IFN-gamma was produced by spleen cells obtained from WT and CD4KO mice on day 3 postinfection, irrespective of alpha -GalCer treatment (data not shown). As shown in Fig. 5, spleen cells from infected WT mice on day 7 after treatment with vehicle did not produce IFN-gamma , except for at the highest dose of antigen, at which low production was detected, while a dose-dependent production of IFN-gamma was observed in spleen cells obtained from alpha -GalCer-treated WT mice. These cells also produced a large amount of IFN-gamma in response to ConA (1 µg/ml) but did not produce any IFN-gamma or only a marginal amount upon stimulation with PPD (1 and 10 µg/ml), which was used as an irrelevant control antigen (data not shown). Furthermore, no significant reduction in their IFN-gamma synthesis was observed after depletion of both NK and NKT cells by magnetic separation with anti-NK1.1 MAb (undepleted, 7,851 ± 1,530 pg/ml; control IgG treated, 5,610 ± 733 pg/ml; anti-NK1.1 MAb-treated, 5,472 ± 886 pg/ml). In contrast, spleen cells obtained from infected CD4KO mice did not produce IFN-gamma even after treatment with alpha -GalCer (Fig. 5).


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  		FIG. 5.   Induction of Th1 cells by alpha -GalCer treatment. WT and CD4KO mice received intraperitoneal injections of alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0 and 3 after intravenous injection of C. neoformans (106/mouse). On day 7, spleen cells were prepared and restimulated with the indicated doses of live fungal organisms for 2 days. Then the concentration of IFN-gamma in the culture supernatants was measured by ELISA. Each bar represents the mean and SD for triplicate cultures. Experiments were repeated three times with similar results. ND, not detected.

To confirm that the development of Th1 cells caused by alpha -GalCer treatment was through activation of NKT cells, treated or untreated spleen cells obtained from WT or NKT-KO mice on day 7 after infection were restimulated with live microorganisms and culture supernatants were assayed for IFN-gamma production. As shown in Fig. 6, spleen cells from alpha -GalCer-treated mice produced a large amount of IFN-gamma upon restimulation with 106 and 107 cells of C. neoformans per ml. In contrast, spleen cells from NKT-KO mice treated with alpha -GalCer produced only little IFN-gamma even when they were restimulated with the highest dose of antigen.


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  		FIG. 6.   NKT-cell-dependent induction of Th1 cells by alpha -GalCer treatment. WT and NKT-KO mice received intraperitoneal injections of alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0 and 3 after intravenous injection of C. neoformans (106/mouse). On day 7, spleen cells were prepared and restimulated with the indicated doses of live fungal organisms for 2 days. Then the concentration of IFN-gamma in the culture supernatants was measured by ELISA. Each bar represents the mean and SD for triplicate cultures. Experiments were repeated twice with similar results. ND, not detected.

Elimination of C. neoformans in the lungs and spleen by treatment with alpha -GalCer. Finally, we examined the effect of alpha -GalCer treatment on the fungal burdens in the lungs and spleen on days 7 and 14 after infection with C. neoformans. As shown in Fig. 7, the number of live microorganisms in the spleen was significantly lower in mice treated with alpha -GalCer at both time points than in control mice. Lung burdens of the microorganism were significantly reduced by the same treatment on day 7 postinfection and became undetectable on day 14, but the number of live colonies was over the detection limit in the vehicle-treated mice.


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  		FIG. 7.   Effect of alpha -GalCer treatment on local host resistance to cryptococcal infection. WT mice received intraperitoneal injections of alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0, 3, and 7 after intravenous injection of C. neoformans (106/mouse). On days 7 and 14, the live colonies in the lungs (A) and spleen (B) were counted. Each bar represents the mean and SD for three mice. Experiments were repeated three times with similar results. Open bars, vehicle-treated mice; solid bars, alpha -GalCer-treated mice; ND, not detected; *, P < 0.05 compared with the vehicle-treated mice.

To elucidate the mechanism of alpha -GalCer-induced host resistance, WT and NKT-KO mice were treated with vehicle or this agent on days 0 and 3 after infection with C. neoformans and the fungal burdens in the lungs and spleen were examined on day 7. As shown in Fig. 8, alpha -GalCer treatment significantly reduced the number of live microorganisms in both organs of WT mice compared to those of mice treated with vehicle only. In contrast, in NKT-KO mice, alpha -GalCer failed to change the fungal count, although obvious differences in the severity of infection was not found between the two strains. Furthermore, we also examined the effect of neutralizing MAb against IFN-gamma to define its role in alpha -GalCer-induced host resistance. As shown in Fig. 9, treatment with this MAb strongly impaired alpha -GalCer-induced eradication of C. neoformans from the lungs and spleen relative to that in mice treated with control rat IgG.


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  		FIG. 8.   alpha -GalCer causes NKT-cell-dependent host resistance to cryptococcal infection. WT and NKT-KO mice were treated with intraperitoneal injections of alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0 and 3 after intravenous injection of C. neoformans (106/mouse). On day 7, the live colonies in the lungs (A) and spleen (B) were counted. Each bar represents the mean and SD for six mice. NS, not significant; *, P < 0.05 compared with vehicle-treated mice.


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  		FIG. 9.   Effect of anti-IFN-gamma MAb on alpha -GalCer-induced host resistance to cryptococcal infection. WT mice were intraperitoneally injected with alpha -GalCer (2 µg/mouse) or the same volume of vehicle on days 0 and 3 after intravenous injection of C. neoformans (106/mouse). The alpha -GalCer-treated mice were injected intraperitoneally with 200 µg of anti-IFN-gamma MAb or control rat IgG on days -1, 0, and 3 after infection. On day 7, the live colonies in the lungs (A) and spleen (B) were counted. Each bar represents the mean and SD for six mice. *, P < 0.05 compared with vehicle-treated mice; NS, not significant; **, P < 0.05 compared with alpha -GalCer-treated and Ab-untreated mice.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The major finding of the present study is that activation of Valpha 14+ NKT cells by treatment with alpha -GalCer resulted in increased production of IFN-gamma in the sera of mice infected with C. neoformans and augmented local host resistance to this infection in the lungs and spleen. This effect of alpha -GalCer was mediated by NKT cells, because the synthesis of this cytokine and protection against cryptococcal infection were not induced in NKT-cell-deficient mice. In addition, neutralization of endogenously synthesized IFN-gamma by a specific Ab canceled most of the protective effect of this agent. These results are similar to those reported recently by Gonzalez-Aseguinolaza et al. (14), who showed that alpha -GalCer-induced activation of Valpha 14+ NKT cells protected mice against liver-stage infection with malaria parasites but not in IFN-gamma -deficient mice. These studies revealed that alpha -GalCer increased the host resistance to cryptococcal infection through production of IFN-gamma . Compatible with this interpretation is the demonstration by other investigators that NKT cells contributed to some Th1-mediated responses including host resistance to Toxoplasma gondii infection (12), granuloma formation caused by mycobacterial cell wall components (1), Salmonella-induced liver injury (18), and elimination of metastatic tumors (9, 24, 36).

The level of IFN-gamma synthesis in the early period after alpha -GalCer administration in infected mice was similar to that in uninfected mice. The data indicate that this response occurred independently of specific antigens and suggest that the source of IFN-gamma production was not T cells but innate immune cells. Compatibly, spleen cells obtained from alpha -GalCer-treated mice on day 3 postinfection did not produce any IFN-gamma upon restimulation with cryptococcal antigens, which indicated that Th1 cells responding to the fungus had not yet been induced at this stage. In further experiments, serum IFN-gamma levels in CD4KO mice were comparable to those in WT mice at the same time intervals, and depletion of NK cells, but not of NKT cells, by anti-ASGM1 Ab partially but significantly inhibited such production. These results indicated that the NK cell is at least one of the producers of IFN-gamma , although the contribution of NKT cell remains unclear. Recent studies from other laboratories (5, 10) demonstrated that NKT cells produced IFN-gamma as early as 2 h after treatment with alpha -GalCer and that activation of NKT cells resulted in a rapid induction of IFN-gamma synthesis by NK cells. Thus, in our study, such cross talk between NKT cells activated by alpha -GalCer and NK cells may operate for early induction of IFN-gamma production.

On the other hand, the cellular source of late-phase IFN-gamma production caused by alpha -GalCer was identified to be CD4+ T cells. In uninfected mice, IFN-gamma could not be detected in serum on day 7 even when the mice were treated with alpha -GalCer, while a large amount of IFN-gamma was produced by this treatment in mice infected with C. neoformans. In addition, no such production was noted in CD4KO mice, indicating that CD8+ T cells did not contribute to this response. These results suggest that alpha -GalCer induced the differentiation of Th1 cells that respond to cryptococcal antigens in a specific manner in infected mice. This interpretation was confirmed by in vitro experiments. Spleen cells obtained from alpha -GalCer-treated mice on day 7 postinfection produced a large amount of IFN-gamma upon restimulation with the fungal antigen, while only a marginal amount was detected in culture supernatants of spleen cells from infected and vehicle-treated mice. These cells produced lower or undetectable amounts of IFN-gamma upon stimulation with PPD than they did in response to the fungal organisms, although all these cells exhibited profound responses to ConA. Furthermore, such IFN-gamma synthesis by spleen cells was completely abrogated both in CD4KO and NKT-KO mice, which is consistent with the idea of alpha -GalCer-induced differentiation of Th1 cells through activation of NKT cells. In an alternative interpretation, CD4+ NKT cells, which are also absent in CD4KO mice, but not CD4+ T cells, might be the major source of this cytokine. However, this is unlikely because spleen cells obtained from infected and alpha -GalCer-treated mice on day 7 produced a comparable level of IFN-gamma even after depletion of NKT cells by magnetic separation with anti-NK1.1 MAb. Taken together, these results indicated that C. neoformans-specific Th1 cells were induced by treatment with alpha -GalCer through activation of NKT cells.

In early studies, Yoshimoto et al. (40) demonstrated that activation of NKT cells by in vivo administration of anti-CD3 MAb resulted in a rapid production of IL-4 and proposed that this population may be the major source of early IL-4 production, which contributes to the differentiation of Th2 cells. Compatibly, Singh et al. (32) showed that activation of NKT cells by alpha -GalCer induced a T-cell response to protein antigen polarized toward the Th2-dominant condition, although both IFN-gamma and IL-4 were acutely produced. However, several investigations subsequently indicated that the Th2 response was not hampered in beta 2-microglobulin- or CD1d-deficient mice, which have markedly reduced numbers of NKT cells. Therefore, the role of these cells in Th2-cell development is controversial. Furthermore, in a recent study by Cui et al. (10), activation of Valpha 14+ NKT cells by alpha -GalCer resulted in the suppression of the Th2 response, such as IgE generation caused by Nippostrongylus brasilliensis, through the production of IFN-gamma . In our unpublished results, levels of IL-4 and IL-13 in serum increased in C. neoformans-infected mice after treatment with alpha -GalCer, with similar kinetics to those observed in IFN-gamma synthesis. At present, the significance of alpha -GalCer-induced Th2 cytokine production during C. neoformans infection remains unclear because Th2 cytokines, such as IL-4 and IL-10, were reported to act as suppressive cytokines in host defense against this microorganism (3, 11, 21).

In conclusion, we demonstrated in the present study that treatment with alpha -GalCer induced IFN-gamma production by innate immune cells including NK cells at the early phase of infection with C. neoformans and Th1 cells at the late phase through activation of Valpha 14+ NKT cells. These changes resulted in improvement of the local host resistance to this infection. These results suggest that this synthetic glycolipid may be a possible candidate agent for immunotherapy of intractable cryptococcosis. For this purpose, however, further investigations are necessary, which include determination of the precise mechanism of alpha -GalCer in the regulation of the Th1-Th2 cytokine balance by NKT cells.


    ACKNOWLEDGMENTS

We thank F. G. Issa (Word-Medex, Sydney, Australia) for critical reading and editing of the manuscript and Mayumi Kinjo for the technical assistance.

This work was supported in part by grants-in-aid for Science Research (C) (09670292 and 12670261) from the Ministry of Education, Science and Culture and by grants from the Ministry of Health and Welfare, Japan.


    FOOTNOTES

* Corresponding author. Mailing address: The First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Phone: 81(98) 895-1144. Fax: 81(98) 895-1414. E-mail: kawakami{at}med.u-ryukyu.ac.jp.

Editor:   S. H. E. Kaufmann


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Infection and Immunity, January 2001, p. 213-220, Vol. 69, No. 1
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.1.213-220.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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