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The Journal of Immunology, 2000, 164: 64-71.
Copyright © 2000 by The American Association of Immunologists

Synergistic Effects of IL-4 and IL-18 on IL-12-Dependent IFN-{gamma} Production by Dendritic Cells1

Taro Fukao, Satoshi Matsuda and Shigeo Koyasu2

Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mouse splenic dendritic cells (DCs) produce IFN-{gamma} in response to IL-12. In the present study, we analyzed effects of Th1 and Th2 cytokines on IFN-{gamma} production by DCs. IL-18 produced by DCs and macrophages acts in an autocrine manner and augments IL-12-induced IFN-{gamma} production by DCs as also observed in T and NK cells. Surprisingly, IL-4, a Th2 cytokine, also acts synergistically with IL-12 on IFN-{gamma} production by DCs. In addition, IL-4 markedly enhances IFN-{gamma} production when DCs are stimulated through CD40 or MHC class II. These results indicate that both Th1 and Th2 cytokines act on DCs during T cell-DC interaction upon Ag presentation. p38 mitogen-activated protein kinase is constitutively activated in mature DCs and is required for IFN-{gamma} production by DCs. IL-18 but not IL-4 or IL-12 further activates the p38 mitogen-activated protein kinase activity, suggesting that IL-4 and IL-18 enhance IFN-{gamma} production through distinct intracellular signal transduction pathways in DCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs)3 derived from bone marrow are specialized in capture, processing, and transport of Ags to lymphoid organs where they activate Ag-specific naive T lymphocytes. Upon Ag capture or microbial infection, DCs produce various cytokines such as IL-12 that act on T lymphocytes (1). There are two potent stimulation pathways to induce IL-12 production by DCs. Microbial infection such as invasion by Toxoplasma gondii induces rapid IL-12 production without T cell interaction (2). A second pathway is dependent on T cells and requires CD40-CD40 ligand (CD40L; CD154) and/or MHC class II-TCR interaction (3, 4, 5). IL-12 production by DCs through CD40-CD40L interaction is thought to be important for Th1 induction (6, 7).

IL-12 acts on T and NK cells to induce the production of IFN-{gamma}. Cumulative evidence has shown the importance of IFN-{gamma} in both innate and acquired immunity (8, 9, 10, 11, 12, 13, 14, 15). It has long been assumed that the only cells producing IFN-{gamma} in response to IL-12 are T and NK cells. However, recent studies have shown that macrophages and B cells are also capable of producing IFN-{gamma} in response to IL-12 (16, 17, 18). Furthermore, we have demonstrated that DCs are also able to produce significant amounts of this cytokine upon Listeria monocytogenes infection or IL-12 administration (19), suggesting the presence of an autocrine-positive feed back pathway in APCs.

Action of IL-12 is influenced by other cytokines such as IL-18. IL-18, originally designated as IFN-{gamma}-inducing factor, strongly augments IFN-{gamma} production by T cells, cytotoxicity of NK cells, and T cell proliferation (20). IL-18 acts synergistically with IL-12 in inducing IFN-{gamma} from T cells undergoing differentiation to Th1 cells as well as committed Th1 cells. IL-18 also induces IFN-{gamma} production by NK cells in both mouse and human (20, 21). Thus, it has been suggested that both IL-12 and IL-18 are required for effective differentiation of Th1 cells. In contrast to IL-12 and IFN-{gamma}, the Th2-type cytokines, IL-4 and IL-10, show inhibitory effects on IFN-{gamma} production by T cells (22, 23). Furthermore, these cytokines are reported to suppress DC functions (1, 4, 24, 25, 26).

Recent studies on molecular mechanisms regulating IFN-{gamma} gene expression in T cells have shown the importance of p38 mitogen-activated protein kinase (MAPK) pathway (27). p38, a member of the MAPK superfamily, is activated by various stimuli, such as proinflammatory cytokines (e.g., IL-1ß and TNF-{alpha}), LPS, and various environmental stresses (heat, osmotic stress, UV irradiation) (28, 29, 30, 31). p38 MAPK has been implicated in the regulation of expression of many cytokine genes (27, 32, 33, 34).

Because various stimuli are able to activate DCs to secrete IL-12 and then IFN-{gamma}, it is of interest to examine the role of other cytokines and the molecular mechanisms of regulation of IFN-{gamma} production by DCs. Here we report synergistic action of IL-4 as well as IL-18 with IL-12 in augmenting IFN-{gamma} production by DCs. In contrast, IL-10 has no effect on the ability of DCs to produce IFN-{gamma}. We further show that p38 MAPK activities are important in IFN-{gamma} production by DCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 mice were purchased from Sankyo Labo Service (Tokyo, Japan). B10.D2-recombinase activating gene (Rag)-2-deficient mice, Rag-2-/- mice that had been backcrossed to B10.D2/nSnJ for 10 generations (Ref. 19 and S.K., unpublished observations), were obtained from Taconic (Germantown, NY). All mice were maintained in specific pathogen-free conditions in our animal facility and used between 6 and 12 wk of age. All experiments were performed in accordance with our Institutional Guidelines.

Abs, cytokines, and reagents

The following mAbs were purchased from PharMingen (San Diego, CA): HL3-FITC, -PE (anti-CD11c); 53-6.7-PE, -biotin (anti-CD8{alpha}); PO3-biotin (anti-CD86); purified C17.8 (anti-mIL-12p40/p70); purified 3/23 (anti-CD40). Anti-mouse I-Ab,k mAb was purchased from Chemicon International (Temecula, CA). Rabbit polyclonal anti-asialoGM1 ({alpha}-ASGM1) Ab was purchased from Wako Pure Chemical Industries (Osaka, Japan). Affinity-purified rabbit anti-p38 MAPK Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant mouse IL-12 was purchased from Sigma (St. Louis, MO). Recombinant mouse IL-18 and anti-mouse IL-18 mAb were purchased from MBL (Nagoya, Japan). Recombinant mouse IL-10 was purchased from Pharma Biotechnologie Hannover (Hannover, Germany). Purified recombinant mouse IL-4 (100 U/ng) expressed in a baculovirus system (35) was a generous gift from A. Miyajima (Tokyo University, Tokyo, Japan). A p38 MAPK-specific inhibitor, SB203580, was obtained from Calbiochem (San Diego, CA). RPMI 1640 supplemented with 10% FCS (HyClone, Logan, UT), 2-ME (50 µM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and sodium pyruvate (1 mM) was used as the complete culture medium (CM).

DC preparation

DCs were prepared from collagenase-digested spleens (Collagenase D; Boehringer Mannheim, Indianapolis, IN), as described previously (19, 36). Briefly, collagenase-digested spleen cells were suspended in a dense BSA solution in PBS (p = 1.080; Sigma), overlaid with 1 ml of FCS-free RPMI 1640 medium, and centrifuged in a swing bucket rotor at 9500 x g for 20 min at 4°C. The cells in a low-density fraction at the interface were collected and washed twice. The cells were resuspended in CM and allowed to adhere to plastic dishes for 2 h. After nonadherent cells were depleted, adherent cells were incubated for an additional 18 h to allow DCs to detach. After this incubation, floating cells were collected and DCs were positively purified using anti-CD11c (N418) MicroBeads and a magnetic cell separation system column (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified cells were routinely >94% CD11c+ I-A+. In some experiments, the nonadherent cell fraction from the overnight incubation procedure was stained with a mixture of the following biotinylated mAbs: anti-CD3{epsilon}, 145-2C11; anti-CD4, GK1.5; anti-B220, RA3-6B2. Cells were then incubated with streptavidin MicroBeads and depleted by magnetic cell separation system (Miltenyi Biotec). After this depletion, purity of DCs was >80%. An additional purification procedure was done to separate DCs into CD8{alpha}+ and CD8{alpha}- subsets. The CD8{alpha}+ subset was positively selected with anti-CD8{alpha} (Ly-2) MicroBeads (Miltenyi Biotec), and, from the negative fraction of this selection, the CD8{alpha}- subset was purified with anti-CD11c (N418) MicroBeads (Miltenyi Biotec).

Flow cytometric analysis

Cells were stained with FITC-, PE-, or biotin-conjugated mAbs in PBS-2% FCS, washed, and analyzed on a FACScan using the CELLQuest program (Becton Dickinson, San Jose, CA). Biotinylated mAbs were detected with streptavidin Red 670 (Life Technologies, Gaithersburg, MD).

p38 kinase assay

Purified DCs (2 x 106 in 1 ml CM) were stimulated with cytokines for indicated time periods and lysed in a lysis buffer solution consisting of 20 mM Tris-HCl, pH 7.4, 12.5 mM ß-glycerophosphate, 2 mM EGTA, 10 mM NaF, 1 mM benzamidine, 1% Triton X-100, 2 mM DTT, 1 mM sodium orthovanadate, 1% aprotinin, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A. The lysates were centrifuged at 15,000 x g for 30 min, and p38 MAPK was immunoprecipitated from the postnuclear supernatant with anti-p38 Ab and protein A-Sepharose beads (Amersham, Arlington Heights, IL). The precipitates were washed twice with a buffer solution consisting of 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20, and once with an extraction buffer solution consisting of 20 mM Tris-HCl, pH 7.4, 12.5 mM ß-glycerophosphate, 2 mM EGTA, 10 mM NaF, 1 mM benzamidine, and 2 mM DTT. Samples were then mixed with 3 µg of His-tagged ATF2 in the presence of 50 µM ATP, 10 mM MgCl2, and 74 kBq of [{gamma}-32P]ATP and incubated at 30°C for 20 min in a final volume of 15 µl. Reactions were terminated by boiling in a Laemmli’s sample buffer solution, resolved by SDS-PAGE, and radioactivities were quantified on an image analyzer, BAS2000 (Fujix, Tokyo, Japan).

Cytokine assays

Titers of IFN-{gamma} in the culture supernatants were determined by Quantikine M ELISA kit (R&D Systems, Minneapolis, MN), mouse IFN-{gamma} ELISA kit (Endogen, Woburn, MA), or mouse IFN-{gamma} ELISA kit, Intertest-{gamma} (Genzyme, Cambridge, MA). It was noted that the titers obtained by Genzyme kits were always one-fifth of those obtained by the other two ELISA kits. Therefore, when Genzyme kits were used in an experiment, it is stated in the figure legend.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-18 synergistically acts on DCs with IL-12 in IFN-{gamma} production

As shown previously (19), mouse splenic DCs produce high levels of IFN-{gamma} in response to IL-12 stimulation (Fig. 1Go, A and B). Because IL-18 strongly augments IFN-{gamma} production by T, B, and NK cells, we examined the effect of IL-18 on IFN-{gamma} production by DCs. Purified DCs were cultured for 3 days in the presence of IL-12 with various concentrations of IL-18, and the amounts of IFN-{gamma} in the culture supernatants were determined by ELISA. As shown in Fig. 1GoC, we found that IL-18 dramatically enhances IL-12-dependent IFN-{gamma} production by DCs in a dose-dependent manner. However, IL-18 alone had little effect on IFN-{gamma} production without IL-12. The same results were obtained with DCs isolated from {alpha}-ASGM1 Ab-treated Rag-2-/- mice (19), indicating that the effect of IL-18 is not due to contaminated lymphocytes (Fig. 1GoD). These results indicate that IL-18 acts on DCs synergistically with IL-12 to produce IFN-{gamma} in a way similar to its action on T, B, and NK cells and macrophages (17, 18, 20).



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  		FIGURE 1. Synergistic effect of IL-18 on IL-12-induced IFN-{gamma} production by isolated DCs. A, DCs were isolated as described in Materials and Methods and stained with biotinylated anti-MHC class II I-A mAb and PE-conjugated anti-CD11c mAb to check the purity on a FACScan (b). Negative control data were obtained by staining with biotinylated and PE conjugated anti-human CD3{epsilon} mAb, Leu-4 (a). Purity of DCs was routinely >94% as determined on the basis of I-A and CD11c expression. B, Purified DCs were cultured (1 x 105/well in 200 µl CM) in the presence of various concentrations of IL-12. After 3 days incubation, titers of IFN-{gamma} in the culture supernatants were determined by ELISA. Results are representative of three independent experiments with consistent results. *, 10 µg/ml anti-IL12 mAb was added in culture to neutralize endogenous IL-12. C, Purified DCs were cultured (5 x 104/well in 200 µl CM) in the presence of IL-12 (1 ng/ml) and various concentrations of IL-18. Anti-IL-12 mAb was added at a final concentration of 10 µg/ml. Supernatants were harvested on day 3 and examined for IFN-{gamma} by ELISA. Results are representative of three independent experiments with consistent results. D, DCs purified from {alpha}-ASGM1 Ab-treated Rag-2-/- mice were cultured (5 x 104/well in 200 µl CM) in the presence of IL-12 (1 ng/ml) and various concentrations of IL-18. Supernatants were harvested on day 3 and examined for IFN-{gamma} by ELISA. Results are representative of two independent experiments with consistent results.

 
Effects of IL-4 and IL-10 on IFN-{gamma} production by DCs

Th2-type cytokines, IL-4 and IL-10, are known to suppress IFN-{gamma} production by T cells (22, 23). Thus, we investigated whether IL-4 and IL-10 also suppress IFN-{gamma} production by DCs. To our surprise, IL-4 dramatically augmented IL-12-induced IFN-{gamma} production by DCs in a dose-dependent manner, whereas IL-4 alone had no effect (Fig. 2GoA). These results indicate that IL-4 also acts synergistically with IL-12 on IFN-{gamma} production by DCs. Although not shown, IL-4 from four different sources were used with consistent results. In contrast to IL-4, IL-10 showed no significant effect. IL-10 neither enhanced nor inhibited IFN-{gamma} production by DCs (Fig. 2GoB). Furthermore, IL-10 showed no effect on the synergistic effect of IL-4 (Fig. 2GoC). Action of IL-4 does not involve IL-18 produced by DCs because addition of anti-IL-18 mAb had no effect on IL-4 action (Fig. 2GoC). These results collectively show that a Th2 cytokine, IL-4, acts synergistically with IL-12 on IFN-{gamma} production by DCs, whereas IL-10, another Th2 cytokine, has no effect.



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  		FIGURE 2. Effects of Th2-type cytokines on IFN-{gamma} production by DCs. A, IL-4 enhances IL-12-dependent IFN-{gamma} production by DCs. Purified DCs were cultured (3 x 104/well in 200 µl CM) with IL-12 (0.5 ng/ml) and various concentrations of IL-4. After 3 days culture, culture supernatants were harvested and measured for IFN-{gamma} by ELISA. Results are representative of four independent experiments with consistent results. B, IL-10 does not have significant effects on IFN-{gamma} production by DCs. DCs (3 x 104/well in 200 µl CM) were incubated in the presence of IL-12 (0.5 ng/ml) and indicated concentrations of IL-10. Culture supernatants were tested for IFN-{gamma} production by a Genzyme ELISA kit after 3 days culture. One of three independent experiments with consistent results is shown. C, IL-10 does not affect IFN-{gamma} production by DCs induced by IL-4 and IL-12. DCs (3 x 104/well in 200 µl CM) were incubated with IL-12 (0.5 ng/ml) in the presence of IL-4 (500 U/ml), IL-10 (20 ng/ml), and/or anti-IL-18 mAb (5 µg/ml). Titers of IFN-{gamma} in the culture supernatants were measured by ELISA after a 3-day incubation. Results are representative of two independent experiments with similar results.

 
Synergistic effect of IL-4 on CD40 and MHC class II induced IFN-{gamma} production by DCs

DCs produce IL-12 upon various stimuli through different mechanisms such as microbial invasion and T cell-DC interaction (2, 3, 4, 5, 37). The latter is dependent on CD40-CD40L (CD154) and/or MHC class II-TCR interaction (3, 4, 5). Thus, it is possible that endogenous IL-12 production resulting from CD40-CD40L interaction or MHC class II-TCR ligation induces IFN-{gamma} production by DCs. To this end, purified DCs were cultured in the presence of anti-CD40 or anti-MHC class II mAbs, and IFN-{gamma} production was examined by ELISA. As expected, both CD40 cross-linking by anti-CD40 mAb and MHC class II cross-linking by anti-MHC class II mAb induced IFN-{gamma} production by DCs (Fig. 3Go, A and B). Addition of neutralizing mAb against IL-12 markedly suppressed IFN-{gamma} production by CD40 or MHC class II cross-linking. Thus, IFN-{gamma} production by CD40 or MHC class II cross-linking is likely dependent on endogenous IL-12 in an autocrine manner. IL-4 was also shown to synergistically induce IFN-{gamma} production by anti-CD40 mAb and anti-MHC class II mAb (Fig. 3Go, C and D). Anti-IL-12 mAb blocked such synergistic effect of IL-4, indicating that IL-4 also favors IL-12-dependent IFN-{gamma} production during DC-T cell interaction.



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  		FIGURE 3. Effect of IL-4 on CD40 or MHC class II cross-linking induced IFN-{gamma} production by DCs. A, IL-12 secreted by CD40 cross-linking induces IFN-{gamma} production by DCs. Purified DCs (2 x 105/well in 200 µl CM) were stimulated with 10 µg/ml anti-CD40 mAb 3/23 in the presence or absence of anti-IL-12 mAb (10 µg/ml). After 3 days incubation, culture supernatants were harvested and analyzed for IFN-{gamma} by ELISA. Results are representative of three independent experiments with consistent results. B, IL-12 secreted by MHC class II cross-linking induces IFN-{gamma} production by DCs. Purified DCs (2 x 105/well in 200 µl CM) were stimulated with 10 µg/ml anti-MHC class II mAb in the presence or absence of anti-IL-12 mAb (10 µg/ml). After 3 days incubation, culture supernatants were harvested and analyzed for IFN-{gamma} by ELISA. Results are representative of two independent experiments with consistent results. C, IL-4 enhances IFN-{gamma} production induced by CD40 cross-linking. DCs (5 x 104/well in 200 µl CM) were stimulated with anti-CD40 mAb (10 µg/ml) in the presence or absence of anti-IL-12 mAb (10 µg/ml) and various concentrations of IL-4. IL-4 was added 1 h before addition of anti-CD40 mAb. After 3 days culture, IFN-{gamma} in the culture was measured by ELISA. One of three independent experiments with consistent results is shown. D, IL-4 enhances IFN-{gamma} production induced by MHC class II cross-linking. DCs (5 x 104/well in 200 µl CM) were stimulated with anti-MHC class II mAb (10 µg/ml) in the presence or absence of anti-IL-12 mAb (10 µg/ml) and various concentrations of IL-4. IL-4 was added 1 h before addition of anti-MHC class II mAb. After 3 days culture, IFN-{gamma} in the culture was measured by ELISA. One of two independent experiments with similar results is shown.

 
IL-4 and IL-18 shows synergism with IL-12 on CD8{alpha}- myeloid as well as CD8{alpha}+ lymphoid DCs in IFN-{gamma} production

In the mouse, two distinct DC populations are present in the spleen. CD8{alpha}+DEC205+CD11b- and CD8{alpha}-DEC205-CD11b+ DC represent lymphoid and myeloid DCs, respectively (38). It has been reported that they differ in their ability to induce Th1/Th2 responses (2, 7, 39, 40). Furthermore, we have previously demonstrated that CD8{alpha}+ DCs are the major IFN-{gamma} producers in response to IL-12 stimulation (19). These results prompted us to compare how IL-4 and IL-18 act on these subsets for IFN-{gamma} production. CD8{alpha}+ and CD8{alpha}- DCs were isolated (Fig. 4GoA) and cultured for 3 days in the presence of IL-12 alone or in combination with IL-4 or IL-18. When cultured with IL-12 alone, CD8{alpha}+ DCs produced significantly higher levels of IFN-{gamma} than CD8{alpha}- DCs did (the ratio of amounts produced was about 5:1) as shown previously (19). Interestingly, when IL-4 or IL-18 are present with IL-12, no difference was observed between two DC subsets in the amounts of IFN-{gamma} production (Fig. 4GoB). The CD8{alpha}- DC subset is thus capable of producing high amounts of IFN-{gamma} in the presence of IL-4 or IL-18 together with IL-12.



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  		FIGURE 4. IFN-{gamma} production by distinct DC subsets. A, Purification of CD8{alpha}+ and CD8{alpha}- DCs. DC subpopulations were purified as described in Materials and Methods. Purity of CD11c+I-A+ DCs was >90%. Purity of CD8{alpha}+ and that of CD8{alpha}- cells among total DCs were 90% and 94%, respectively. B, IFN-{gamma} production by distinct DC subpopulations. Both CD8{alpha}+ and CD8{alpha}- DCs (2 x 104/well in 150 µl CM) were cultured in the presence of IL-12 (1 ng/ml) alone, IL-12 plus IL-4 (1000 U/ml), and IL-12 plus IL-18 (20 ng/ml). After 3 days incubation, IFN-{gamma} production was measured by ELISA. Results are representative of two independent experiments with consistent results.

 
p38 MAPK activity is involved in IFN-{gamma} production by DCs and augmented by IL-18 but not by IL-4

A MAPK superfamily member, p38 MAPK, is implicated in regulating the expression of various cytokine genes (27, 32, 33, 34). It has recently been reported that p38 MAPK is required for IFN-{gamma} production in Th1 cells (27). Thus, we examined whether p38 MAPK is involved in the IFN-{gamma} production by DCs. Purified DCs were stimulated with IL-12 alone or in combination with IL-4 or IL-18, and p38 MAPK activities were measured. As shown in Fig. 5GoA, activity of p38 MAPK was readily detected in nonstimulated DCs, and IL-12 did not increase the p38 MAPK activity. When DCs were stimulated with a combination of IL-12 and IL-18, a 2-fold increase in p38 MAPK activity was observed. In contrast, IL-4 had no effect on p38 MAPK activity. These results imply that p38 MAPK is constitutively activated in mature DCs at a low level, and IL-18 but not IL-12 or IL-4 augments its activity.



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  		FIGURE 5. p38 MAPK activity is required for IFN-{gamma} production by DCs and is enhanced by IL-18. A, p38 MAPK activity in response to various stimulation. Purified DCs (2 x 106 in 1 ml CM) were stimulated with IL-12 (10 ng/ml) for 15 min or 30 min, IL-12 plus IL-4 (1000 U/ml) for 15 min, or IL-12 plus IL18 (10 ng/ml) for 15 min at 37°C and the activities of p38 MAPK were measured as described in Materials and Methods. As a positive control, DCs were incubated with 10 µg/ml anisomycin for 40 min. p38 MAPK activity is shown as the autoradiograph of the phosphorylated ATF2 (upper panel) and relative incorporation of [32P] in each column. Aliquots of total lysates were subjected to Western blotting analysis to confirm the equal loading (lower panel). B, Inhibition of IFN-{gamma} production by the drug SB203580. Purified DCs (5 x 104/well in 150 µl CM) were stimulated with (a) IL-12 (1 ng/ml), (b) IL-12 plus IL-4 (500 U/ml), or (c) IL-12 plus IL-18 (1 ng/ml) in the presence of different concentrations (0.1–10 µM) of SB203580 or vehicle (DMSO) alone (0 µM). After 30 h incubation, titers of IFN-{gamma} present in the culture supernatants were measured by ELISA. One of two independent experiments with consistent results is shown.

 
We next analyzed the effect of SB203580, a specific inhibitor of p38 MAPK (41), to examine the involvement of p38 MAPK in the production of IFN-{gamma} by DCs. As shown in Fig. 5GoB, a, SB203580 inhibited IFN-{gamma} production in a dose-dependent manner when DCs were stimulated with IL-12. Similarly, SB203580 inhibits IFN-{gamma} production by DCs stimulated by IL-12 in combination with IL-4 or IL-18 (Fig. 5GoB, b and c). The presence of SB203580 did not affect the viability of the cells when culture supernatants were harvested (data not shown). From these results, we conclude that the basal activity of p38 MAPK in mature DCs is required for IL-12-induced IFN-{gamma} production.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-18 was cloned as an IFN-{gamma}-inducing factor (20, 21), which with IL-12 synergistically activates IFN-{gamma} production in T, B, and NK cells (17, 42, 43, 44). We show here that IL-18 also acts synergistically with IL-12 for IFN-{gamma} production by DCs. In T and B cells, up-regulation of IL-18 receptor expression by IL-12 stimulation is one prevailing explanation for the synergism of IL-12 and IL-18 on IFN-{gamma} induction (44, 45). Mature DCs constitutively produce small amounts of IL-12 and anti-IL-12 mAb completely blocks basal level of IFN-{gamma} production by DCs (Fig. 1GoB). We found that DCs express IL-18 receptor mRNA as detected by RT-PCR without exogenous IL-12 stimulation (our unpublished observation). It is possible that endogenous IL-12 production results in the expression of IL-18 receptors on DCs. Thus activation of DCs by phagocytosis or microbial infection likely leads to the production of higher amounts of IL-12, which further up-regulate IL-18 receptor expression.

As shown here, DCs can produce IFN-{gamma} in response to IL-12 and IL-18, both of which are produced by DCs (46). These results suggest a novel role of DCs in innate and acquired immunity. The importance of rapid IFN-{gamma} production has been shown in innate immunity such as in the immediate response to L. monocytogenes and T. gondii infection (2, 8, 11, 13, 19). Based on our results and recent reports, we propose a model of acute IFN-{gamma} production in the innate immune response (Fig. 6GoA). In response to pathogens such as T. gondii, IL-12 is produced by DCs independently from T cells (2) and triggers an IL-12 autocrine pathway and IFN-{gamma} production by DCs (19, 37). IFN-{gamma} subsequently activates macrophages to produce IL-12 and IL-18 (20, 47, 48), both of which act on DCs and macrophages to further induce IFN-{gamma} production (18). Such a positive feedback cycle results in amplification of IFN-{gamma} in the innate immune response. As demonstrated here, IL-18 also acts on DCs and macrophages in an autocrine manner for IFN-{gamma} production. In addition, IFN-{gamma} derived from DCs likely influences Th1 induction in acquired immunity by acting on T cells during Ag presentation (9, 10). Recent reports provide strong evidence for the importance of DCs in driving Th1 and Th2 responses (7, 39, 40, 49). IL-18 presumably acts on Th1 induction as a potent enhancer of IFN-{gamma} production by DCs.



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  		FIGURE 6. A model of the acute IFN-{gamma} production cycle in the innate and acquired immune responses. A, A model of the acute IFN-{gamma} production cycle in the innate immune response. B, IFN-{gamma} production by DCs during DC-T cells interaction.

 
One of surprising results is the synergism of IL-4 with IL-12 on IFN-{gamma} production by DCs. IFN-{gamma} and IL-4 are typical cytokines produced by Th1 and Th2 cells, respectively, and suppress the differentiation of Th2 and Th1, respectively (15, 50). Our findings clearly contradict the general view that IL-4 suppresses IFN-{gamma} production. However, it should be noted that several groups have previously observed phenomena that are consistent with our findings. First, experimental autoimmune uveoretinitis is a typical Th1-dependent disease. In contrast to the expectation, IL-4-treated rats in an experimental autoimmune uveoretinitis model showed an aggravation of symptoms, and the amounts of IFN-{gamma} production were actually augmented by IL-4 treatment (51). Such enhanced production of IFN-{gamma} could be due to either direct activation of DCs by IL-4 or Th1 activation by DCs exposed to IL-4. Second, in an Ag-induced arthritis model in mice, treatment with neutralizing anti-IL-4 mAb inhibited the disease, suggesting that IL-4 plays a proinflammatory role and can act as a mediator of Th1 development in this system (52). Third, in addition, it has recently been reported that IL-4 is required for the development of a Th1 response to Candida albicans (53); the IFN-{gamma} production by CD4+ Th cells was reduced in IL-4-deficient mice in response to a virulent strain of C. albicans. Based on our results, it is likely that IL-4 acts on DCs during Ag presentation and induces IFN-{gamma} production followed by efficient Th1 induction in these cases.

Lingnau et al. (54) demonstrated a novel alternative pathway of Th1 induction. IL-4 in combination with TGF-ß favors Th1 development, and this process requires IFN-{gamma} (54). Our findings that IL-4 up-regulates IFN-{gamma} production by DCs strongly support this novel Th1-inducing pathway. The role of DCs in such a Th1 induction pathway is not only as APCs but also the source of IFN-{gamma} in the presence of IL-4. We also showed that IL-4 enhances IFN-{gamma} production by DCs upon CD40 or MHC class II cross-linking, which mimics DC-T cell interaction (Figs. 3Go and 6GoB). In this pathway, endogenous IL-12 production by DCs seems essential because addition of anti-IL-12 mAb blocked IFN-{gamma} production to near basal levels. Because IFN-{gamma} is indispensable for the alternative pathway, DCs are the most likely candidates as the source of IFN-{gamma} in this pathway.

It has been shown that DCs can be divided into distinct subsets and that different subsets of DCs induce different Th subsets (7, 39, 40, 49). In mice, it has been reported that CD8{alpha}+ lymphoid DCs induce the Th1-type response, whereas CD8{alpha}- myeloid DCs trigger the Th2-type response in vivo (7, 39, 40). We have previously demonstrated that IL-12 induces IFN-{gamma} production predominantly by CD8{alpha}+ lymphoid DCs rather than CD8{alpha}- myeloid DCs (19), which is consistent with the notion that CD8{alpha}+ lymphoid DCs play a critical role in Th1 induction. We found in the present study that CD8{alpha}- myeloid DCs are also capable of producing high amounts of IFN-{gamma} when IL-4 or IL-18 coexists with IL-12, suggesting that both DC subsets produce IFN-{gamma} when infected with microorganisms and induced to produce IL-18. In studies reporting that the distinct DC subsets differed in their capacity to induce Th subsets, CD8{alpha}+ and CD8{alpha}- DCs were separately isolated in vitro and reconstituted to in vivo after Ag-pulse (7, 39, 40). Although it is likely that CD8{alpha}+ lymphoid DCs and CD8{alpha}- myeloid DCs have intrinsic characteristics for induction of Th1 and Th2 responses, respectively, pathogens are likely presented to T cells by both DC subsets in actual infection. Various other factors involved in Ag presentation by DCs during induction of Th subsets must be investigated to understand the molecular mechanisms of Th1/Th2 differentiation.

In this study, the role of p38 MAPK in IFN-{gamma} production by DCs was also investigated. Involvement of p38 MAPK has been implicated in IFN-{gamma} production by Th1 cells (27). We found that the specific inhibitor of p38 MAPK, SB203580, suppresses IFN-{gamma} production by DCs in response to IL-12 alone or in combination with IL-4 or IL-18, suggesting the importance of p38 MAPK in IFN-{gamma} production by DCs. p38 MAPK activity is readily observed in isolated DCs and is not enhanced by IL-12 (Fig. 5Go). Rescigno et al. (55) reported that MAPK family members c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase were activated during LPS-induced maturation of an immortalized immature mouse splenic DC line. Because DCs present in the spleen are in an immature stage and induced to maturate during isolation procedures by unknown mechanisms (1, 56), it is likely that p38 MAPK is already activated and its activity is maintained in mature DCs. The fact that p38 MAPK activity is unaltered by IL-12 yet SB203580 inhibits IL-12-induced IFN-{gamma} production indicates that the basal activity of p38 MAPK in mature DCs is required for the IL-12 signaling. Although IL-4 and IL-18 synergistically induced IFN-{gamma} production, only IL-18 augments p38 MAPK activity. This finding suggests that the increase in p38 MAPK activity is one of the mechanisms of the synergistic effect of IL-18 for IL-12-induced IFN-{gamma} production by DCs. The mechanism of synergistic effect of IL-4 is different from that of IL-18 and remains to be examined. Because IL-4 acts on both mouse and human bone marrow- or monocyte-derived DCs induced by GM-CSF (57, 58), it is possible that IL-4 modulates some maturation or growth state of DCs during culture, influencing the capacity of DCs to respond to IL-12.

In summary, we demonstrate synergistic effects of both Th1- and Th2-type cytokines, namely IL-4 and IL-18, with IL-12 on IFN-{gamma} production by DCs. We also demonstrate a role for p38 MAPK pathways in IL-12-induced IFN-{gamma} production by DCs. Biochemical analyses show that IL-4 and IL-18 enhance the effects of IL-12 through distinct intracellular signaling pathways. Such synergistic effects of IL-4 and IL-18 are of interest in understanding the role of DCs in both innate and acquired immunity, especially in the Th1 vs Th2 balance.


   Acknowledgments
 
We are grateful to Dr. A. Miyajima for recombinant IL-4, Drs. T. Ohteki, H. Suzuki, K. Suzue, L. K. Clayton, D. M. Frucht, and J. J. O’Shea for valuable discussion and advice, M. Fujiwara, C. Maki for help in experiments, and A. Sakurai for excellent animal care. T.F. thanks A. Ohshima and Sanshiro Fukao for careful reading of the manuscript.


   Footnotes
 
1 This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan (10153261), a Keio University Special Grant-in-Aid for Innovative Collaborative Research Project, and a grant from the Japan Society for the Promotion of Science (JSPS-RFTF 97L00701). Back

2 Address correspondence and reprint requests to Dr. Shigeo Koyasu, Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: Back

3 Abbreviations used in the paper: DC, dendritic cell; CM, culture medium; MAPK, mitogen-activated protein kinase; Rag, recombinase activation gene; CD40L, CD40 ligand. Back

Received for publication August 19, 1999. Accepted for publication October 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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