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An Absolute Requirement for STAT4 and a Role for IFN- as an Amplifying Factor in IL-12 Induction of the Functional IL-18 Receptor Complex¹

Masakiyo Nakahira^*, Michio Tomura^*, Masayuki Iwasaki^*, Hyun-Jong Ahn, Yang Bian^*, Toshiyuki Hamaoka^*, Tsunetaka Ohta, Masashi Kurimoto and Hiromi Fujiwara^2,*

^* Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Osaka, Japan; Department of Microbiology, Kyun-Hee University Medical School, Seoul, Korea; and Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-12 and IL-18 are both proinflammatory cytokines that contribute to promoting Th1 development and IFN- expression. However, neither IL-12R nor IL-18R is expressed as a functional complex on most resting T cells. This study investigated the molecular mechanisms underlying the induction of an IL-18R complex in T cells. Resting T cells expressed IL-18R chains but did not exhibit IL-18 binding sites as detected by incubation with rIL-18 followed by anti-IL-18 Ab, suggesting a lack of IL-18R expression in resting T cells. Although they also failed to express IL-12R, stimulation with anti-CD3 plus anti-CD28 generated IL-12R. Exposure of these cells to IL-12 led not only to up-regulation of IL-18R expression but also to induction of IL-18R binding sites on both CD4⁺ and CD8⁺ T cells concomitant with IL-18R mRNA expression. The IL-18 binding site represented a functional IL-18R complex capable of exhibiting IL-18 responsiveness. IL-12 induction of an IL-18R complex and IL-18R mRNA expression was not observed in STAT4-deficient (STAT4^-/-) T cells and was substantially decreased in IFN-^-/- T cells. However, the failure of STAT4^-/- T cells to induce an IL-18R complex was not corrected by IFN-. These results indicate that STAT4 and IFN- play an indispensable role and a role as an amplifying factor, respectively, in IL-12 induction of the functional IL-18R complex.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin 18 is a proinflammatory cytokine that stimulates NK and T cells and functions to enhance innate immunity as well as specific Th1 immune responses (1, 2). IL-18 was originally described as a factor capable of inducing IFN- production by primed T cells (3). IL-18 acts directly on NK cells to stimulate IFN- synthesis (4) and up-regulate killing activity (4, 5). In contrast, this cytokine does not exert direct effects on T cells. In synergy with IL-12, IL-18 has been shown to induce strikingly high levels of IFN- production by T cells (6, 7, 8) and to enhance Th1 development (9, 10).

IL-18 responsiveness depends on the expression of the IL-18R complex as shown by IL-18-binding capacity. Initial studies (8) have shown that IL-18 binding sites are not detected on either resting or anti-CD3/anti-CD28-stimulated (TCR-triggered) T cells. However, IL-18 binding sites were induced on TCR-triggered lymph node T cells (8) and on a Th1 clone (7) when exposed to IL-12. The capacity of IL-12 to induce IL-18 binding sites was regarded as a mechanism for the synergy between IL-12 and IL-18. Thereafter, two subunits of IL-18R, IL-1R-related protein (now renamed IL-18R) and accessory protein-like molecule (AcPL,³ now renamed IL-18R), were characterized (11, 12). Both IL-18R subunits belong to the IL-1R family (12, 13) and resemble components of the IL-1R complex. IL-18R (IL-1R-related protein) represents a low-affinity receptor for IL-18 (11). Like IL-1RAcPL, IL-18R (IL-18RAcPL) fails to bind IL-18, but with IL-18R forms the IL-18R complex that represents the IL-18 binding site and functions as the IL-18R signaling complex (12, 14). Several groups have recently investigated the effect of IL-12 on the induction/up-regulation of IL-18R and expression (15, 16, 17, 18). The results obtained in these studies appear controversial in terms of its differential dependence on IL-12. Moreover, the molecular mechanism underlying IL-12 induction/up-regulation of IL-18R and/or expression remains unclear.

In the present study, we investigated the effect of IL-12 on IL-18R and gene expression and cell surface expression of the IL-18R complex. We show that IL-12 acts on TCR-triggered T cells to generate the IL-18R complex through inducing IL-18R mRNA expression along with up-regulation of IL-18R expression. The results also demonstrate that TCR-triggered T cells from STAT4-deficient (STAT4^-/-) mice fail to express IL-18R mRNA and to up-regulate IL-18R expression after IL-12 stimulation. Consequently, the IL-18R complex was not detected on STAT4^-/- T cells. The requirement for STAT4 in the induction of the IL-18R and IL-18R complex by IL-12 was not due simply to a lack of IFN- expression in STAT4^-/- T cells because exogenous IFN- failed to correct the defect in STAT4^-/- T cells. Moreover, IL-12 still induced moderate albeit reduced levels of the IL-18R complex in IFN-^-/- T cells. These results illustrate that STAT4 plays an indispensable role in IL-12 induction of the functional IL-18R and IFN- functions as an amplifying factor for this IL-12 effect.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

BALB/c mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). STAT4^-/- BALB/c mice (BALB/c-Stat4^tm1Gru) (19) and IFN--deficient (IFN-^-/-) BALB/c mice (BALB/c-Ifng^tm1Ts) (20) were obtained from The Jackson Laboratory (Bar Harbor, ME). These knockout mice were bred in our laboratory and used at 6–9 wk of age.

Reagents

Murine rIL-12 and murine rIL-18 were provided from Genetics Institute (Cambridge, MA) and Hayashibara Biochemical Laboratories (Okayama, Japan), respectively. Anti-CD3 (145-2C11) (21), anti-CD28 (Pv-1) (22), anti-I-A^d/b (34-5-3S) (23), anti-CD4 (American Type Culture Collection, Rockville, MD; ATCC clone GK1.5), anti-CD8 (ATCC clone 2.43), and anti-murine IL-12 (C17.8) (24) mAbs were purified from culture supernatants or ascitic fluids of the respective hybridomas. Rat anti-mouse IL-18R mAb and anti-murine IL-18 polyclonal Ab were provided by Hayashibara Biochemical Laboratories. Allophycocyanin (APC)-conjugated anti-CD4 and FITC-conjugated anti-CD8 mAbs were obtained from BD PharMingen (San Diego, CA). Biotinylated mouse anti-rat IgG and biotinylated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch (West Grove, PA). PE-conjugated streptavidin was obtained from BD Biosciences (Mountain View, CA). Normal rat IgG was purchased from BioMeda (Foster City, CA).

Preparation of T cell subsets

Lymph node cells were depleted of B cells and Ia⁺ APC by immunomagnetic-negative selection as follows: cells were allowed to react with anti-I-A^d/b mAb and then incubated with magnetic particles bound to goat anti-mouse Ig (Advanced Magnetics, Cambridge, MA). A T cell population depleted of anti-I-A^d/b-labeled and surface Ig⁺ cells was obtained by removing cell-bound magnetic particles with a rare earth magnet (Advanced Magnetics). Purity of the resulting T cell populations was examined by flow cytometry and found to be consistently >95%.

Stimulation of T cells with anti-CD3 plus anti-CD28 mAb and subsequent exposure to IL-12

T cell cultures were done as previously described (8). Briefly, anti-CD3 (5 µg/ml) and anti-CD28 mAbs (2 µg/ml) were coimmobilized to individual wells of 24-well culture plates (Corning 2582; Corning Glass Works, Corning, NY) in a volume of 0.5 ml in PBS. After 3 h, solutions were discarded and plates were washed with PBS twice. Purified T cells were cultured in 2 ml of RPMI 1640 medium supplemented with 10% FBS and 2-ME at 1.5 x 10⁶ cells/well in a humidified atmosphere of 5% CO₂ at 37°C. Cells were harvested 48 h later and after washing, recultured in the presence of 1000 pg/ml rIL-12 for an additional 48 h.

Measurement of IFN- concentrations

IFN- concentrations were determined by ELISA as previously described (8).

Immunofluorescence and flow cytometry

For the detection of IL-18R, cells were incubated with anti-IL-18R mAb for 20 min at 4°C. Cells were then incubated with biotinylated mouse anti-rat IgG followed by PE-conjugated streptavidin. For the detection of CD4/CD8 expression, cells were stained directly with APC-conjugated anti-CD4 or FITC-conjugated anti-CD8 mAb. The detection of IL-12R was performed as previously described (25). Briefly, cells were incubated with 7.5 ng of rIL-12 in 40 µl of medium for 60 min at 4°C. Cells were washed and then incubated with 1 µg of rat anti-mouse IL-12 mAb (C17.8) for 30 min at 4°C. After washing, cells were allowed to react with 0.1 µg of biotinylated mouse anti-rat IgG followed by incubation with PE-conjugated streptavidin. The staining protocol used in detection of IL-12R was applied to the detection of IL-18R as previously described (8). Cells were incubated with 40 ng of rIL-18 in 40 µl of medium for 60 min at 4°C. Cells were washed and then incubated with 0.1 µg of rabbit anti-mouse IL-18 polyclonal Ab for 30 min at 4°C. After washing, cells were allowed to react with 0.1 µg of biotinylated goat anti-rabbit IgG followed by incubation with PE-conjugated streptavidin. Stained cells were analyzed with a FACSCalibur (BD Biosciences). In some experiments, IL-12 or IL-18 binding sites or IL-18R was detected by gating on a CD4⁺ or CD8⁺ population.

RT-PCR

Total RNA was prepared from cytokine-stimulated T cells by the acid guanidium-thiocyanate-phenol-chloroform method. Total RNA (1 µg) was reverse transcribed into cDNA in a total volume of 20 µl using random primers and Superscript II RNase H^- Reverse Transcriptase (Life Technologies, Rockville, MD). PCR amplification was conducted in a total volume of 50 µl of 1x PCR buffer (Takara Shuzo, Otsu, Japan) containing 1.0 µl of the first-strand cDNA, 0.25 mM of each dNTP, 2 µM of each primer, and 2.5 U of Taq DNA polymerase (Takara Shuzo). The following oligonucleotides were used: IL-18R sense primer 5'-CTGAAGGATGCCGAGTTTGGAGATGAGGGC-3'; IL-18R antisense primer 5'-CACTATACACACTGCTGCCACAGAGGCGAG-3'; IL-18R sense primer 5'-GGCTCCATTCATTGTCCCAGTCTCAGCTGC-3'; IL-18R antisense primer 5'-CCGTGTTGTGTTCCCAATGGAGTTCTGGGC-3'; -actin sense primer 5'-AGAAGAGCTATGAGCTGCCTGACG-3', and -actin antisense primer 5'-CTTCTGCATCCTGTCAGCAATGCC-3'. Cycle parameters were: annealing 1 min at 60°C (IL-18R) or 55°C (-actin), elongation 2 min at 72°C, and denaturation 1 min at 94°C. Resulting PCR products were separated in 1% agarose gel and visualized by ethidium bromide staining. Sequences of the IL-18R, IL-18R, and -actin (for standardization), were amplified out of each cDNA batch with 24, 24, and 23 amplification cycles, respectively.

Preparation of cell lysates and nuclear extracts

Nuclear extracts were prepared essentially as described previously (26), except that the following buffers were used. After washing with PBS, cells were resuspended in cell lysis buffer (20 mM HEPES-NaOH (pH 7.9), 20 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM EDTA, and 0.1 mM EGTA) supplemented with 0.2% Nonidet P-40 (NP40), 1 mM DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.1 mM Pefablock (Boehringer Mannheim, Mannheim, Germany). The nuclei were pelleted and then extracted with vigorous agitation at 4°C in the above buffer without NP40 but containing 0.42 M sodium chloride, 20% glycerol, and protease inhibitors as above.

EMSA

The binding reaction was performed in a total volume of 20 µl in the following buffer: 10 mM HEPES-NaOH (pH 7.9), 1 mM EDTA, 30 mM NaCl, 0.1% NP40, 1 mM DTT, 1 mg/ml BSA, and 5% glycerol. Each reaction, also containing 2 µg of poly(dI-dC) and ³²P end-labeled probe, was initiated by the addition of 8 µg of nuclear extract and allowed to incubate at room temperature for 30 min before electrophoretic analysis on a 5.25% polyacrylamide gel in 0.25x TBE (Tris-borate-EDTA) buffer. The NF-B consensus oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGG-3'; Ref. 27) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Resting T cells express IL-18R but do not exhibit IL-18-binding capacity

Purified lymph node T cells were examined for the expression of IL-12R and IL-18R. Our previous study (8) showed that resting T cells do not exhibit IL-12 binding sites as detected by incubation with rIL-12 followed by staining with anti-IL-12 Ab. This is confirmed in Fig. 1 (upper left). Fig. 1 (upper center) also shows that resting T cells fail to exhibit IL-18 binding sites as the IL-18R complex. However, they express considerable levels of an IL-18R component, the IL-18R chain (Fig. 1, upper right).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Resting T cells express IL-18R but not IL-12 or IL-18 binding sites. Purified resting T cells from WT, STAT4-/-, or IFN--/- BALB/c mice were stained for IL-12 or IL-18 binding sites as described in Materials and Methods. Cells were also stained with anti-IL-18R mAb.

Purified T cells were stimulated with anti-CD3 plus anti-CD28 mAb for 48 h. As shown in Fig. 2 (upper panels), the resultant TCR-triggered T cells, both CD4 and CD8, exhibited IL-12 binding sites. However, the IL-18 binding site was still undetectable in TCR-triggered T cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Induction of IL-12 but not of IL-18 binding sites by TCR triggering. Purified T cells from WT, STAT4-/-, or IFN--/- BALB/c mice were stimulated with anti-CD3 and anti-CD28 mAbs for 48 h in 24-well culture plates. Cells were stained for CD4, CD8, and IL-12 binding sites. Analysis gates for IL-12 binding sites were set on the whole population, CD4+ cells, and CD8+ cells. Cells were also stained for IL-18 binding sites.

IL-12 stimulation after TCR triggering induces the IL-18 binding site along with up-regulation of IL-18R expression

T cells that expressed IL-12R after TCR triggering were cultured in the presence of IL-12 for 48 h. IL-12 stimulation generated IL-18 binding sites in both CD4⁺ and CD8⁺ T cells although the frequency of positive cells was higher in the latter than in the former (Fig. 3, upper panels). We also examined whether IL-12 stimulation up-regulates IL-18R expression in TCR-triggered T cells. As shown in Fig. 4 (upper panels), exposure of TCR-triggered CD4⁺ and CD8⁺ T cells to IL-12 resulted in considerable up-regulation of IL-18R expression.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Failure of STAT4-/- T cells to express IL-18 binding sites after IL-12 exposure. Purified T cells were stimulated with anti-CD3/anti-CD28 for 48 h. Cells were then cultured with 1000 pg/ml IL-12 for 48 h and examined for the expression of IL-12 binding sites on the whole, CD4+, and CD8+ populations.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Failure of STAT4-/- T cells to up-regulate IL-18R expression after IL-12 exposure. IL-12-exposed T cells were stained doubly for IL-18R and CD4 or CD8.

STAT4 is the most critical of IL-12 signaling molecules (19, 28, 29, 30). We determined whether STAT4 deficiency affects the IL-12-mediated induction of the IL-18R complex. This was done using T cells from STAT4^-/- mice as well as cells from IFN-^-/- mice because a representative of IL-12 bioactivities is the capacity to stimulate IFN- production (31, 32), and this capacity depends upon STAT4 activation (19, 30). Resting T cells from STAT4^-/- and IFN-^-/- mice, like those from wild-type (WT) mice, exhibited neither IL-12 nor IL-18 binding sites but expressed the IL-18R chain (Fig. 1, middle and lower panels). Moreover, the induction of the IL-12 binding site after TCR triggering was not affected by STAT4 or IFN- deficiency (Fig. 2, middle and lower panels). However, stimulation of TCR-triggered STAT4^-/- T cells with IL-12 failed to induce the IL-18 binding site (Fig. 3, middle panels) and to up-regulate IL-18R expression (Fig. 4, middle panels). Although IFN-^-/- T cells expressed IL-18 binding sites as well as the IL-18R chain after IL-12 exposure, the levels were substantially reduced in CD4⁺ T cells and in a part of CD8⁺ T cells as compared with those observed in WT T cells.

The failure of IFN- to correct the defect in STAT4^-/- T cells

To determine whether the defective induction of the IL-18 binding site in STAT4^-/- T cells results mainly or partly from their failure to produce IFN-, rIFN- was included in IL-12-stimulated cultures. As shown in Fig. 5, addition of rIFN- to the IL-12 stimulation cultures of IFN-^-/- T cells up-regulated the induction of the IL-18 binding site and IL-18R expression to levels comparable to those observed in WT T cells stimulated with IL-12 in the absence or presence of rIFN-. In contrast, the same amount of rIFN- failed to exert any effect on the induction of the IL-18 binding site and up-regulation of the IL-18R chain in STAT4^-/- T cells. These results indicate that IFN- functions as an amplifying factor for up-regulation of IL-18 binding sites including the IL-18R chain. However, the requirement for STAT4 in the induction of the IL-18 binding site involves a mechanism that is distinct from that of IL-12 induction of STAT4-mediated IFN- expression.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. IFN- cannot correct the failure of STAT4-/- T cells to induce IL-18 binding sites and to up-regulate IL-18R expression. Anti-CD3/anti-CD28-stimulated WT, STAT4-/- , and IFN--/- T cells were cultured with IL-12 (1000 pg/ml) alone or in combination with IFN- (10 ng/ml) for 48 h.

IL-12 induces IL-18R mRNA expression along with up-regulation of IL-18R mRNA expression in a STAT4-dependent manner

The results of Figs. 3 and 4 suggest that IL-18R mRNA is only marginally expressed in resting and TCR-triggered WT T cells and induced to detectable levels after IL-12 exposure and that IL-18R induction does not occur in STAT4^-/- T cells. To test this assumption, we determined mRNA expression levels of IL-18R in parallel with those of IL-18R by RT-PCR. Fig. 6 shows the time course of IL-18R and -chain gene expression and of the cell surface expression of the IL-18 binding site in WT T cells after exposure to IL-12. IL-18R mRNA was detectable before IL-12 exposure and was up-regulated after the exposure. In contrast, IL-18R mRNA was hardly detectable before the exposure but following IL-12 stimulation was gradually induced (Fig. 6A). In much closer association with the time course of IL-18R than with that of IL-18R mRNA expression, the IL-18 binding site became detectable on IL-12-exposed WT T cells (Fig. 6B).

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Time course of IL-18R and mRNA expression and induction of IL-18 binding sites after IL-12 stimulation. Anti-CD3/anti-CD28-stimulated WT T cells were cultured with IL-12 for the indicated times (h). A, Total RNA was isolated at each time point and subjected to RT-PCR for IL-18R, IL-18R, and -actin (control) mRNA transcripts. B, Activated T cells were harvested at each time point and stained for IL-18 binding sites.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. Effects of IL-12 on IL-18R and mRNA expression in TCR-triggered T cells. Total RNA was isolated from freshly prepared resting T cells and T cells exposed to IL-12 after TCR triggering. These were subjected to RT-PCR for IL-18R, IL-18R, and -actin (control) mRNA transcripts.

Differential levels of NF-B activation following IL-18 stimulation in WT, STAT4^-/-, and IFN-^-/- T cells

We investigated the correlation between IL-18R expression and IL-18R-mediated signaling. For this, the activation of NF-B by IL-18 stimulation was examined in WT, STAT4^-/-, and IFN-^-/- T cells exposed to IL-12 following TCR triggering. Fig. 8 illustrates that IL-18 stimulation induced high levels of NF-B activation in IL-12-exposed WT T cells expressing the functional IL-18R complex, which is consistent with previous results (10). In contrast, NF-B activation was markedly reduced in STAT4^-/- T cells expressing only an IL-18R component. Although IFN-^-/- T cells expressed apparently reduced levels of the functional IL-18R complex (Fig. 3), NF-B activation in these T cells was only slightly decreased compared with WT T cells.

View larger version (54K): [in this window] [in a new window]   FIGURE 8. NF-B activation in WT, STAT4-/-, and IFN--/- T cells exposed to IL-12 following TCR triggering. TCR-triggered WT, STAT4-/-, and IFN--/- T cells were cultured with IL-12 for 48 h. IL-12-treated T cells were stimulated with IL-18 for 30 min. Nuclear extracts were prepared from these IL-18-stimulated T cells and examined for binding to an oligonucleotide probe corresponding to a consensus NF-B-binding sequence in EMSA.

IFN- production by TCR-triggered T cells and T cells exposed to IL-12 after TCR triggering

We examined the capacity of TCR-triggered and TCR-triggered IL-12-exposed T cells to produce IFN- in response to IL-12 and/or IL-18 (Fig. 9). Stimulation of TCR-triggered WT T cells with IL-12 produced an appreciable amount of IFN- (Fig. 9A). Although IL-18-stimulated IFN- production by these T cells was weak, the combined stimulation with IL-12 and IL-18 synergistically enhanced IFN- production. In contrast, the same combined stimulation of STAT4^-/- T cells induced reduced levels of IFN- production compared with those produced by WT T cells. These levels were comparable to those of IL-18-stimulated WT and STAT4^-/- T cells. The results in Fig. 9B show that IL-18 responsiveness leading to IFN- production is generated in IL-12-exposed T cells from WT, but not from STAT4^-/- mice. These data are consistent with the results on induced expression of the IL-18 binding site (Figs. 2 and 3) and support the notion that IL-12 plays an indispensable role in the STAT4-dependent induction of the functional IL-18R complex.

View larger version (33K): [in this window] [in a new window]   FIGURE 9. Failure of STAT4-/- T cells to produce large amounts of IFN- in response to IL-12 plus IL-18. Purified T cells from WT and STAT4-/- mice were stimulated with anti-CD3 plus anti-CD28 (TCR triggering) for 48 h. These TCR-triggered T cells were stimulated with IL-12, IL-18, or a combination of these cytokines for 24 h (A). B, TCR-triggered T cells were cultured in the presence or absence of 1000 pg/ml IL-12 for 48 h. IL-12-exposed and unexposed T cells were stimulated with IL-18 for 24 h. Culture supernatants were assayed for IFN- concentrations by ELISA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our present data show that resting T cells express IL-18R mRNA and its protein on their cell surfaces, whereas IL-18R mRNA is hardly detected in resting T cells. After TCR triggering, when IL-12R⁺ T cells are further stimulated with IL-12, IL-18R transcripts become detectable along with up-regulation of IL-18R mRNA expression. In correlation with IL-18R mRNA induction, IL-12-exposed T cells express the IL-18R complex represented by an active IL-18 binding site and become responsive to IL-18 stimulation. Because neither IL-18R up-regulation nor IL-18R induction occurs in STAT4^-/- T cells, both of these events are totally dependent on IL-12-mediated STAT4 activation. Thus, IL-18 responsiveness in T cells depends on the capacity of IL-12-activated STAT4 to induce/up-regulate IL-18R components, in particular, to induce the IL-18R chain that is negligibly expressed in resting T cells.

A number of recent studies have addressed the question of whether IL-12 is required to induce/up-regulate either of the IL-18R components or both (15, 16, 17, 18), and whether STAT4 plays a role in the mediation of the IL-12 bioactivity (18). Our results concur with some of the observations made in these studies and appear discordant with others. Because the above studies themselves produced seemingly controversial results, our present results should be discussed in terms of whether the observations made in all of these studies can be compromised. Concerning the effect of IL-12 on mRNA expression of IL-18R components, a much stronger up-regulation of IL-18R than IL-18R mRNA expression was shown in some reports (15, 17). In contrast, potent up-regulation of IL-18R mRNA expression was observed in other studies using human T cells (16) and a human NK cell line (18). In fact, the study of Sareneva et al. (16) demonstrated that whereas human T cells express marginal levels of IL-18R and mRNAs even after activation (TCR triggering), stimulation with IL-12 strongly enhanced the mRNA expression for both chains. In our mouse model, while mRNA expression for both subunits was up-regulated after IL-12 stimulation, IL-18R mRNA was found to be constitutively expressed in resting T cells at considerable levels. Consistently, resting T cells exhibited surface expression of this subunit. In mouse T cells, it appears that IL-12 contributes more to IL-18R expression than to IL-18R up-regulation.

The discrepancy may be explained by considering the type of target cells tested. In the study of Yoshimoto et al. (15), freshly prepared mouse T cells, without TCR triggering, were directly stimulated with a strikingly high concentration (20 ng/ml) of IL-12 for as long as 72 h to detect IL-18R and mRNA expression. However, most resting T cells do not express IL-12R and a functional IL-12R complex is induced after stimulation of the TCR and CD28 (8, 33, 34, 35). Such TCR-triggered T cells express both IL-12R subunits and respond rapidly to IL-12 at concentrations of 100-1000 pg/ml that are approximately two orders lower than those used in the study of Yoshimoto et al. (15). Our present system may more likely represent the effect of IL-12 on the expression of both IL-18R subunits in recently TCR-triggered mouse T cells.

It may also be important to take into consideration the time points when mRNA expression of IL-18R and was determined following IL-12 stimulation. For example, induction/up-regulation of the IL-18R transcript was not observed 4 h after IL-12 stimulation (Ref. 17 and this study). The study of Kim et al. (18) and ours demonstrate that although up-regulation of IL-18R expression is observed at relatively early time points, IL-18R mRNA up-regulation becomes detectable later (later than 8 h after IL-12 stimulation). It should be, thus, noted that there exists a substantial time difference between the appearance of the effects induced by IL-12 on mRNA expression of the two IL-18R subunits. These observations could also reconcile the discordant results obtained from various studies regarding the effect of IL-12 on IL-18R mRNA expression.

A more important aspect of the present findings concerns the molecular basis for the requirement of IL-12 in the induction of the IL-18R complex. IL-12 signals via the Janus kinase-STAT signal transduction pathway and activates STAT4 and STAT3 through tyrosine kinase 2 and Janus kinase 2 phosphorylation (28, 29, 36). In particular, the role of STAT4 has been well appreciated in the mediation of IL-12 bioactivities including Th1 development and IFN- production of T cells (19, 30). Lawless et al. (17) showed that STAT4 regulates multiple components of IFN--inducing signaling pathways. IL-18R was also included in these. IL-18R should also be included because our present results demonstrated that IL-12-mediated STAT4 activation is an absolute requirement for IL-18R induction in addition to up-regulation of IL-18R expression. Thus, the induction/up-regulation of IL-18R components depends on STAT4 activation.

As a result, STAT4^-/- T cells exposed to IL-12 following TCR triggering failed to express the IL-18R complex as the IL-18 binding site. However, these T cells exhibited detectable albeit markedly reduced levels of IL-18 signaling as evaluated by NF-B activation when compared with WT T cells. This may be related to the fact that they still express IL-18R chains. Nevertheless, these STAT4^-/- T cells failed to produce IFN- in response to IL-18. Therefore, it is obvious that the functional IL-18R complex is not induced in STAT4 deficiency.

Despite clear-cut evidence for the requirement of STAT4 (Ref. 17 and this study), it is unknown how STAT4 functions to up-regulate/induce mRNA expression of the IL-18R components. STAT4 is required for IL-12-induced IFN- expression (19, 30). The mechanism for STAT4-induced up-regulation of IL-18R mRNA expression is not just via IFN- production because exogenous IFN- failed to correct the defects observed in STAT4^-/- T cells. Although IFN- functions to amplify the expression of IL-18R and chains, the effect of this cytokine is manifested depending on the presence of STAT4. Thus, it is reasonable to assume that STAT4 functions as a direct transcription factor for IL-18R and gene expression or functions to induce the expression of other genes whose products contribute to the expression of IL-18R genes. Considering the time difference between IL-12-induced IL-18R and mRNA expression, it is possible that STAT4 is required for the expression of IL-18R genes in different manners. Further studies are required to characterize the regulatory element for IL-18R and genes and to determine whether there exist elements with which STAT4 interacts directly or indirectly.

IL-18R was found to be expressed on Th1 but not on Th2 cells (37), indicating a Th1-selective cell surface marker. Moreover, IL-18 signals were shown to promote Th1 development (9, 10). Although IL-18 signals exert their effects on Th1 activation independently of IL-12 signals (9), IL-12 allows IL-18 to signal by inducing a functional receptor. Along with the role of IFN- as an amplifier in the induction of the IL-18R complex, IL-12 induction of the IL-18R complex represents the formation of a positive regulatory loop within the synergy between IL-12 and IL-18 for Th1 development and IFN- production.

IFN- is a representative of inflammatory cytokines. This effector cytokine is produced most efficiently by NK cells in innate immunity and by CD4⁺ and CD8⁺ T cells in acquired immunity through stimulation with the proinflammatory cytokines IL-12 and IL-18. This study also showed that the expression of the IL-18R complex is induced on CD8⁺ T cells via a STAT4-dependent mechanism following IL-12 exposure. Because IL-18-stimulated IFN- production is much stronger in CD8⁺ than in CD4⁺ T cells (8), IL-18R induction on CD8⁺ T cells strengthens IFN- production in acquired immunity. In addition, there may exist additional mechanisms for the synergy between IL-12 and IL-18 for IFN- production by NK and T cells, including mechanisms for the synergy in the signal transduction pathways induced by these two cytokines. Such an elucidation could contribute to our understanding of the overall roles of IL-12 and IL-18 in host defense mechanisms including innate and acquired immunity.

	Acknowledgments

 
We are grateful to Dr. Mark Micallef for critical reviewing of this manuscript and to Mari Yoneyama and Mami Yasuda for secretarial assistance.

	Footnotes

 
¹ This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

² Address correspondence and reprint requests to Dr. Hiromi Fujiwara, Department of Oncology Biomedical Research Center, Osaka University Medical School, 2-2, Yamada-oka, Suita, Osaka 565-0871. E-mail address: hf{at}ongene.med.osaka-u.ac.jp

³ Abbreviations used in this paper: AcPL, accessory protein-like molecule; APC, allophycocyanin; NP40, Nonidet P-40; WT, wild type.

Received for publication February 20, 2001. Accepted for publication May 25, 2001.

	References

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