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The Journal of Immunology, 1999, 162: 5070-5077.
Copyright © 1999 by The American Association of Immunologists

IL-18 Is a Potent Coinducer of IL-13 in NK and T Cells: A New Potential Role for IL-18 in Modulating the Immune Response1 ,2

Tomoaki Hoshino, Robert H. Wiltrout and Howard A. Young3

Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, Frederick Cancer and Research Development Center, Frederick, MD 21702


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-13 and IL-4 have similar biological activities and are characteristic of cytokines expressed by Th2 cells. In contrast, IL-12 and IL-18 have been shown to be strong cofactors for Th1 cell development. In this study, we found strong induction of IL-13 mRNA and protein by IL-2 + IL-18 in NK and T cells. In contrast, IL-12 did not enhance the IL-13 production induced by IL-2 alone. Moreover, IL-13 mRNA and protein expression induced by IL-2 + IL-18 in purified NK and T cells obtained from IFN-{gamma} knockout (-/-) mice were greater than seen in purified cells from normal controls. In contrast, IL-10 production induced by IL-2 and/or IL-12 was not significantly different in IFN-{gamma} (-/-) mice and normal controls. These results suggest IL-13 expression induced by IL-2 + IL-18 may be regulated by IFN-{gamma} in vivo, while IL-10 expression may be IFN-{gamma}-independent. Thus, depending upon the cell type, IL-18 may act as a strong coinducer of Th1 or Th2 cytokines. Our findings suggest that IL-12 and IL-18 have different roles in the regulation of gene expression in NK and T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ and CD8+ Th cells are divided into at least three subsets, Th1, Th2, and Th0, based on cytokine production patterns in response to Ags. Th1 cells mainly produce IL-2 and IFN-{gamma}, while Th2 cells were initially reported to produce IL-4, IL-5, IL-6, IL-10, and IL-13. Th0 cells can produce a combination of the cytokines characteristic of Th1 and Th2 cells, i.e., IL-2, IL-4, IL-13, and IFN-{gamma}, as well as other cytokines (1, 2, 3, 4).

IFN-{gamma} regulates a variety of immunological responses in both innate and acquired immunity. It is the predominant cytokine during Th1-dominated immune reactions. IFN-{gamma} is secreted from T cells and NK cells stimulated with Ags or mitogens (5, 6). It has been reported that IL-1, IL-2, IL-12, IL-18, and TNF-{alpha} are potent inducers and coinducers of IFN-{gamma} in NK and T cells (5, 6, 7, 8, 9, 10, 11, 12, 13). In particular, one of the key events during innate immune reaction is thought to be IL-12 production by macrophages (7). IL-12 then strongly induces NK and T cells to express IFN-{gamma} and GM-CSF mRNA, and is the key cytokine driving Th1 cell differentiation (7).

IL-18 was originally discovered as an IFN-{gamma}-inducing factor, and the structural gene for this protein has recently been cloned (10). IL-18 acts as a strong coinducer of IFN-{gamma} and GM-CSF production in T cells, NK cells, B cells, and macrophages (9, 10, 11, 12, 13). IL-18 also augments NK activity and Fas ligand expression in T cells and NK cells (9, 10, 11, 12, 13). Although IL-18 itself cannot induce strong IFN-{gamma} expression, IL-18 fully induces IFN-{gamma} production in synergy with IL-12 (9, 10, 11, 12, 13). IL-18 is thought to reduce Th2 cytokine (IL-10) production via IFN-{gamma} induction (14). In contrast to IL-12, IL-18 itself cannot induce Th1 differentiation, but potentiates IL-12-driven Th1 development (11, 12). Furthermore, recent studies have demonstrated that IL-12 can up-regulate IL-18 receptor expression in murine Th1 cell clones and purified T and B cells (15, 16). Thus, it is clear that IL-12 can potentiate IL-18 functions. Taken together, IL-12 and IL-18 are thought to be strong inducers or cofactors of Th1 cell development.

IL-13 was initially described as a protein preferentially produced by activated mouse Th2 cells (4). IL-13 acts on normal and malignant B cells, monocytes, macrophages, NK cells, polymorphonuclear cells, osteoblasts, endothelial cells, fibroblasts, and keratinocytes (4). IL-13 has only a low degree of sequence homology with IL-4, but shares most, but not all, of its biological functions with IL-4. IL-13 and IL-4 both induce IgG4 and IgE production by human B cells and up-regulate CD23, CD71, and MHC class II expression on monocytes and B cells (17). IL-13 and IL-4 also down-regulate NK and monocyte function, including cytokine gene expression (18, 19). Recent studies in our laboratory have demonstrated that IL-13 and IL-4 can induce specific STAT6 protein DNA complexes after treatment of human NK cells with these cytokines (19). Moreover, both IL-4 and IL-13 receptor complexes share common signal transducing components (IL-4R{alpha} and IL-13R{alpha}) (20). In contrast, IL-4R, but not IL-13R, complex uses the IL-2R{gamma} chain, and different ligand-binding sites were also observed (4, 20). In addition to being produced by T cells, EBV-transformed B cells and mast cells are able to express IL-13 (21). While NK cells are known to be potent producers of IFN-{gamma}, GM-CSF, TNF-{alpha}, IL-5, and IL-10 (5, 6, 22, 23), we have reported recently that human and mouse NK cells can produce IL-13 in response to IL-2 (24).

In this study, we demonstrate that IL-18 is a potent coinducer of IL-13 production in both NK and T cells. IL-13 mRNA and protein was strongly induced by IL-2 + IL-18 in murine NK and T cells. In T cells and NK cells purified from IFN-{gamma} knockout (-/-) mice, IL-2 + IL-18 induced more IL-13 mRNA and protein synthesis than observed in cells obtained from normal controls. In contrast, we found that in IFN-{gamma} (-/-) mice, IL-10 production by IL-2, IL-12 and/or IL-18 was not significantly different from that observed in normal controls. Thus, the signaling pathways leading to IL-13 and IL-10 gene expression appear to be distinct. The potential importance of Th1 and Th2 type cytokine balance in response to cytokine stimulation in vivo is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

All cell culture was performed utilizing RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Reagents

Recombinant human (h)4 IL-2 (rhIL-2) was obtained from Hoffmann-La Roche (Nutley, NJ). Recombinant mouse (m) IL-12 was generously provided by Genetics Institute (Cambridge, MA), and rmIL-18 was obtained from Pepro Tech (Rocky Hill, NJ). rhIL-1ß was obtained from Biological Resources Branch, National Cancer Institute-Frederick Cancer and Research Development Center (NCI-FCRDC; Frederick, MD). Purified anti-mouse CD3{epsilon} (145-2C11) mAb was purchased from PharMingen (San Diego, CA) and was used for cell culture. PE-conjugated-anti-mouse NK1.1 (PK136), PE-anti-mouse DX5 (DX5), CyChrome-anti-mouse CD3{epsilon} (145-2C11), and FITC-, PE-, or CyChrome-conjugated isotype-matched Ig for FACS analysis were purchased from PharMingen.

Mice

C57BL/6 (B6), SCID, RAG-2 knockout (-/-) (B6 background) (25), and B6 IFN-{gamma} (-/-) mice (26) were used in this study. SCID and IFN-{gamma} (-/-) mice were backcrossed more than 10 generations with B6 mice. These mice were maintained under specific pathogen-free conditions and used for experiments at 8–12 wk of age.

In vivo treatment of mice with IL-2

We used repeated administration of IL-2 to generate large numbers of murine NK and T cells in the liver and spleen (27). B6, IFN-{gamma} (-/-), SCID, and RAG-2 (-/-) mice were injected with 6 x 105 IU rhIL-2 twice a day for 3 days (36 x 105 IU/total/mouse). On day 4, livers and spleens were harvested.

Spleen cell isolation

Spleens were harvested, and single cell suspensions were made by passing the spleens through a metal mesh screen. RBC were lysed in water, and cells were washed and resuspended in 10% FBS RPMI 1640. The cell suspension was then passed through prewetted nylon wool column to deplete B cells and macrophages. Nylon wool nonadherent cells were collected, washed, and resuspended in 10% FBS RPMI 1640 for additional experiments. For further purification, these cells were sorted by MoFlo (Cytomation, Fort Collins, CO) using CyChrome-conjugated anti-mouse CD3 or PE-anti-DX5 mAb, as previously described (28).

Liver cell isolation

Perfused livers were harvested as previously described (29). Single cell suspensions from the livers were made using a Stomacher 80 (Tekmar, Cincinnati, OH). The cell suspension was passed through sterile gauze with HBSS, and cells were washed and resuspended with HBSS. The cell suspension was passed through a Cell Strainer (Becton Dickinson Labware, Franklin Lakes, NJ) with a 100-µm nylon filter and then washed with HBSS. Using Lympholyte-M (Cedarlane Laboratories, Ontario, Canada), mononuclear cells were isolated by the density gradient centrifugation method. Isolated cells were washed with HBSS two times and resuspended in 10% FBS RPMI 1640 for additional experiments.

mRNA assays

Total RNA was isolated using a single-step phenol/chloroform extraction procedure (Trizol; Life Technologies, Gaithersburg, MD). For the RNase protection assay (RPA), 2.5–5 µg of total cytoplasmic RNA was analyzed using the RiboQuant kit (PharMingen) and [33P]UTP-labeled riboprobes, as described by the manufacturer. For Northern blot analysis, 10 µg of total cellular RNA was analyzed following electrophoresis on a 0.8% formaldehyde agarose gel and hybridized with random-primed 32P-labeled cDNA probes as reported (6). mRNA level was quantitated by a densitometer (Molecular Analyst; Bio-Rad Laboratories, Hercules, CA). Mouse GAPDH or chicken ß action was used as the control for the quantitation.

Cytokine assays

Isolated splenic and liver NK and T cells were treated with different stimuli (as indicated in Results) for the specified periods of time, and cell-free supernatants were collected and assayed for cytokine production by sandwich ELISA. The specific ELISA kits utilized were: mouse IFN-{gamma} (R&D Systems, Minneapolis, MN), IL-4 (R&D Systems), IL-10 (R&D Systems), IL-13 (R&D Systems), and IL-5 (Endogen, Woburn, MA). The sensitivity limits were 2 pg/ml, 2 pg/ml, 4 pg/ml, 1.5 pg/ml, and 5 pg/ml, respectively.

Surface Ag analysis by flow cytometry

Three-color analysis was performed using a FACSort (Becton Dickinson) flow cytometer as previously reported (30). Anti-mouse CD16/CD32 mAb (2.4G2; PharMingen) was used to block the nonspecific binding. FITC-, PE-, or CyChrome-conjugated isotype-matched Ig were used for a control in all FACS analysis. A total of 30,000 cells was analyzed in each experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-13 induction by IL-2 + IL-18

We have recently found that human and murine NK cells could produce IL-13 in response to IL-2 (24). Utilizing IFN-{gamma} (-/-) mice, we have observed that, in the absence of IFN-{gamma}, IL-13-producing NK and T cells predominated in vivo (24). It is known that three ILs (IL-1, IL-12, and IL-18) can be potent producers or coinducers of IFN-{gamma} and GM-CSF gene expression in combination with IL-2 in NK cells, as well as T cells (7, 8, 9, 10, 11, 12, 13). Thus, we investigated whether murine NK cells and T cells can produce IL-13 in response to IL-1ß, IL-12, or IL-18. It is known that NK cells in liver and spleen are ~10% and 1–2%, respectively, of the total lymphocytes present in these organs (27). Therefore, we used the phenomenon of IL-2-induced leukocyte rebound to generate larger numbers of murine NK cells in the liver and spleen from in vivo IL-2-treated mice (27). Then, lymphocytes were isolated from the liver and spleen, as described in Materials and Methods. Fig. 1Go shows the relative purity of isolated spleen and liver lymphocytes in a representative experiment. Isolated liver and spleen lymphocytes were stimulated in vitro with IL-2, IL-1ß, IL-12, IL-18, alone or in combination for 3 h. Initially, cytokine production was analyzed with a multiprobe RPA (data not shown). RPA analysis showed that IL-13 mRNA was induced after IL-2 stimulation, but not by IL-1ß, IL-12, nor IL-18 alone in cells obtained from both IFN-{gamma} (-/-) and B6 mice. However, IL-13 mRNA was strongly expressed when cells were treated with the combination of IL-2 + IL-18, but not when treated with IL-2 + IL-1ß, IL-2 + IL-12, IL-12 + IL-1ß, IL-1ß + IL-18, or IL-12 + IL-18 at the 3-h time point. Based on these results, we investigated IL-13 and IFN-{gamma} mRNA expression in cells purified from B6 and IFN-{gamma} (-/-) mice by Northern blot analysis. Spleen cells were isolated from IL-2-treated mice, as described in Materials and Methods, and mRNA was analyzed following treatment with IL-2 (100 U/ml) and/or IL-18 (50 ng/ml) for 3 h (Fig. 2Go). In both B6 and IFN-{gamma} (-/-) mice, IL-13 and IFN-{gamma} mRNA were not strongly expressed without stimulation. IFN-{gamma} mRNA was rapidly and strongly expressed in response to IL-2 + IL-18 in cells isolated from B6 mice. However, IL-13 mRNA was rapidly expressed in response to IL-2 + IL-18 in cells obtained from IFN-{gamma} (-/-) mice. Although IL-13 mRNA was observed in total RNA extracted from IL-2 + IL-18-stimulated spleen cells from B6 mice as analyzed by RPA (data not shown), IL-13 mRNA was barely detectable in the same total RNA when analyzed by Northern blot. In IFN-{gamma} (-/-) mice, IL-13 mRNA levels induced by IL-2 + IL-18 were 3.5- and 2.6-fold higher than in B6 mice when compared with ß-actin and GAPDH mRNA levels, respectively. Similar results were obtained with RPA analysis (data not shown). No significant IL-4 mRNA or protein induction was observed in these cells following treatment with IL-2 + IL-18 as assayed by Northern blot, RPA, and ELISA (data not shown). In addition, no detectable IL-13 mRNA induction by IL-2 + IL-18 was observed in cells obtained from untreated IFN-{gamma} (-/-) and B6 mice when assayed by Northern blot analysis (data not shown).



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  		FIGURE 1. Representative staining pattern of spleen and liver lymphocytes in C57BL/6, B6 IFN-{gamma} (-/-), and B6 RAG-2 (-/-) mice. Spleen and liver lymphocytes were isolated from untreated or in vivo IL-2-treated mice as described in Materials and Methods. Cells were stained with CyChrome-conjugated anti-CD3 and PE-anti-NK1.1 mAb. The number of isolated total spleen lymphocytes from untreated C56BL/6, IFN-{gamma} (-/-), and RAG-2 (-/-) mice were ~1 x 108, 1 x 108, and 2–3 x 107 cells per spleen, respectively. In vivo IL-2-treated spleen lymphocytes were ~1.5 x 108, 1.5 x 108, and 3–5 x 107 cells per spleen, respectively. Untreated liver lymphocytes were ~1 x 106, 1 x 106, and 4 x 105 cells per liver, respectively. In vivo IL-2-treated liver lymphocytes were ~8 x 106, 8 x 106, and 5 x 106 cells per liver, respectively.

 


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  		FIGURE 2. IL-2 + IL-18 induce IL-13 mRNA in spleen lymphocytes isolated from IFN-{gamma} (-/-) mice. Spleen cells were isolated from in vivo IL-2-treated IFN-{gamma} (-/-) and C57BL/6 mice and passed through a prewetted nylon wool column to deplete B cells and macrophages, as described in Materials and Methods. Cells were then stimulated with anti-CD3 (2C11, 5 µg/ml), IL-2 (100 U/ml), IL-18 (50 ng/ml), and IL-2 (100 U/ml) + IL-18 (50 ng/ml) for 3 h. Ten micrograms of total RNA were used for the analysis of IL-13 and IFN-{gamma} mRNA expression by Northern blot analysis.

 
Next, we wished to confirm that spleen cells from IFN-{gamma} (-/-) and B6 mice also could produce IL-13 protein. Spleen cells were isolated from IL-2-treated or -untreated B6 or IFN-{gamma} (-/-) mice, as described in Materials and Methods. Isolated cells were incubated (2 x 106/ml) for 18 h in 10% FBS RPMI 1640 with different stimuli, and the culture supernatants were analyzed by ELISA. Results from one of four independent experiments are shown in Fig. 3Go. As expected, IFN-{gamma} protein was not detected in the supernatants from spleen cells isolated from IFN-{gamma} (-/-) mice. No significant IL-13 production (<10 pg/ml) was found in the supernatants of spleen cells from untreated B6 and IFN-{gamma} (-/-) mice. However, spleen cells from untreated B6 mice synergistically produced IFN-{gamma} in response to IL-2 + IL-18, as previously reported (9, 10). In contrast to untreated mice, IL-13 protein induction was found in spleen cells from both IL-2-treated B6 and IFN-{gamma} (-/-) mice after anti-CD3, IL-2, or IL-2 + IL-18 stimulation. In IL-2-treated IFN-{gamma} (-/-) mice, IL-13 protein levels produced by spleen cells following stimulation with IL-2 or IL-2 + IL-18 in vitro were higher than the levels detected in spleen cell culture supernatants from IL-2-treated normal B6 mice. IL-18 alone induced low levels of IL-13 protein and IL-2 + IL-18 induced much more IL-13 protein than IL-2 alone when different doses of the cytokines were tested (Table IGo). It is clear that IL-18 has a synergistic activity (i.e., more than additive) to induce IL-13 production in combination with IL-2.



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  		FIGURE 3. IL-13 protein expression in spleen lymphocytes. IFN-{gamma} (-/-) and C57BL/6 mice were treated in vivo with IL-2 or untreated for 3 days. Spleen cells were isolated from these mice, passed through a prewetted nylon wool column to deplete B cells and macrophages, as described in Materials and Methods, and were stimulated with anti-CD3 (2C11, 5 µg/ml), IL-2 (100 U/ml), IL-18 (50 ng/ml), and IL-2 (100 U/ml) + IL-18 (50 ng/ml) for 18 h at 2 x 106 cells per ml. The cell-free supernatants were harvested, and cytokines were measured by ELISA. No significant levels of IFN-{gamma} were found in the supernatants from IFN-{gamma} (-/-) mice. The figures show the data from one representative experiment. The ELISA values have an error range of within 10%.

 

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  		Table I. IL-18 has a synergistic activity to induce IL-13 and IFN-{gamma} protein in combination with IL-21

 
We found that IL-13 mRNA levels in total RNA extracted from liver lymphocytes stimulated with IL-2 alone or IL-2 + IL-18 were higher than those seen in spleen cells obtained from IL-2-treated IFN-{gamma} (-/-) mice and control B6 mice when assayed by RPA (data not shown). Therefore, we investigated cytokine levels in the supernatants of lymphocytes obtained from livers of IL-2-treated IFN-{gamma} (-/-) and B6 mice. Results from one of four independent experiments are shown in Fig. 4Go. Four different experiments demonstrated that the levels of IL-13 produced by liver lymphocytes stimulated by IL-2 or IL-2 + IL-18 were higher than those in spleen cells purified from IL-2-treated B6 and IFN-{gamma} (-/-) mice. Moreover, IL-13 protein levels in the supernatants of liver and spleen lymphocytes obtained from IFN-{gamma} (-/-) mice were higher than those levels detected in cells isolated from B6 mice (Figs. 3Go and 4Go). Thus NK and T cells migrating to the liver following in vivo IL-2 treatment appear to have an enhanced ability to express IL-13 when compared with similar cell populations in the spleen.



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  		FIGURE 4. A, IL-13 and IL-10 protein expression in liver lymphocytes. IL-13 but not IL-10 protein expression is synergistically induced by IL-2 + IL-18. B, IL-10 but not IL-13 protein expression is synergistically induced by IL-2 + IL-12. IFN-{gamma} (-/-), C57BL/6 and RAG-2 (-/-) mice were treated in vivo with IL-2 for 3 days. Then liver lymphocytes were isolated from these mice, as described in Materials and Methods, and were stimulated with anti-CD3 (2C11, 5 µg/ml), IL-2 (100 U/ml), IL-18 (50 ng/ml), and IL-2 (100 U/ml) + IL-18 (50 ng/ml) for 18 h at 2 x 106 cells/ml. The cell-free supernatants were harvested, and cytokines were measured by ELISA. No significant levels of IFN-{gamma} were found in the supernatants from IFN-{gamma} (-/-) mice. The data presented in A and B are derived from one of four seperate experiments. The figures show the data from one representative experiment. The ELISA values have an error range of within 10%.

 
IL-10 but not IL-13 induction by IL-2 + IL-12

We investigated whether IL-2 and IL-18 stimulation can induce IL-10 production in lymphocytes obtained from B6 and IFN-{gamma} (-/-) mice. No enhanced IL-10 induction by IL-2 + IL-18 was found in liver and spleen lymphocytes isolated from IL-2-treated IFN-{gamma} (-/-) mice. However, IL-18 weakly induced IL-10 production in liver and spleen cells isolated from IL-2-treated B6 mice in combination with IL-2 (Fig. 4GoA, and data not shown).

It is known that IL-12 and IL-2 + IL-12 can induce both IL-10 and IFN-{gamma} production in T and NK cells (7, 22). In addition, it has been reported that the combination of IL-12 + IL-18 strongly induces IFN-{gamma} production in T cells, NK cells, macrophages, and B cells (9, 10, 11, 12, 13). We investigated whether IL-12, IL-2 + IL-12, and IL-12 + IL-18 could induce IL-10 and IL-13 production in NK and T cells obtained from B6 and IFN-{gamma} (-/-) mice. Although IL-12 did not induce IL-13 production in synergy with IL-2, IL-2 + IL-12 strongly induced IL-10 production in liver and spleen lymphocytes from both B6 and IFN-{gamma} (-/-) mice. In contrast, while IL-12 + IL-18 weakly induced IL-10 and IL-13 production in both B6 or IFN-{gamma} (-/-) mice, IFN-{gamma} production was strongly induced in NK and T cells purified from B6 mice (Fig. 4GoB, and data not shown).

IL-13 production from NK is higher than from T cells

As described above, the IL-13 production induced by IL-2 + IL-18 from liver lymphocytes was much higher than that in spleen cells in both IL-2-treated B6 and IFN-{gamma} (-/-) mice. As shown in Fig. 1Go, the CD3- NK1.1+ NK subset was ~10–20% and 50–70% of spleen and liver lymphocytes in these mice, respectively, while the CD3+ NK1.1+ NK-T subset was ~15–20% and 15–25%, respectively, as previously reported (24). Recent studies have suggested that the NK-T (CD3+ NK1.1+) subset can be a potential producer of the Th2 cytokine, IL-4 (31, 32, 33). Therefore, we speculated that both NK and/or NK-T cells could also produce IL-13 in response to IL-2 + IL-18.

Initially, we used SCID mice (B6 background) that lack T and B cells. RPA and ELISA analysis revealed that isolated liver and spleen lymphocytes isolated from IL-2-treated SCID mice expressed IL-13 mRNA and protein in response to IL-2 + IL-18 (data not shown). However, it has been reported that old SCID mice have a small number of T cells (25). Therefore, in this study, we utilized RAG-2 knockout mice that lack T and B cells to evaluate if NK cells can produce IL-13 in response to IL-2 + IL-18. CD3- NK1.1+ NK cells represented ~30–40% of spleen and liver lymphocytes in untreated RAG-2 (-/-) mice. After 3 days of IL-2 treatment in vivo, >90% of the lymphocytes were CD3- NK1.1+ NK cells in both the spleen and liver (Fig. 1Go). There was no significant population of CD4+, CD8+, TCR{alpha}ß+, and TCR{gamma}{delta}+ cells in these RAG-2 (-/-) mice (data not shown). ELISA and RPA revealed that the liver and spleen NK cells from IL-2-treated RAG-2 (-/-) mice produced IL-13 protein and mRNA in response to IL-2 + IL-18 (Fig. 4GoA, and data not shown). IL-12, IL-2 + IL-12, and IL-12 + IL-18 induced IL-10 protein and mRNA in the purified liver and spleen NK cells from IL-2-treated RAG-2 (-/-) mice. However, no enhanced IL-13 production was observed under these culture conditions (Fig. 4GoB, and data not shown).

Next, we further purified spleen NK and T cells to verify whether NK and T cells in IFN-{gamma} (-/-) mice can produce IL-13 in response to IL-2 + IL-18. Spleen cells were isolated from IFN-{gamma} (-/-) and B6 mice and were sorted using anti-CD3 and anti-DX5 mAb to purify CD3- NK cells and DX5- T cells as described in Materials and Methods. Highly purified (>95%) CD3- NK and DX5- T cells were obtained. A representative result of this study is shown in Fig. 5Go. In B6 and IFN-{gamma} (-/-) mice, both NK and T cells produced IL-13 in response to IL-2 + IL-18. Repeated experiments revealed that in B6 and IFN-{gamma} (-/-) mice, greater amounts (~3- to 10-fold) of IL-13 protein were observed in the supernatants of IL-2 + IL-18-activated spleen NK cells (CD3-, DX5+) than seen in spleen T cells (DX5-, CD3+) (Fig. 5Go, and data not shown). In addition, greater amounts of IL-13 protein were observed in the supernatants of IL-2 + IL-18-activated NK and T cells isolated from IFN-{gamma} (-/-) mice than seen in equivalent cells isolated from B6 mice.



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  		FIGURE 5. Purified NK and T cells synergistically produce IL-13 protein in response to IL-2 + IL-18. After in vivo treatment with IL-2, CD3- NK and DX5- T cells were highly purified from the nylon wool column passed spleen cells by flow cytometry (MoFlo; Cytomation) using CyChrome-conjugated anti-CD3 or PE-DX5 mAb. These purified NK and T cells were stimulated with IL-2 (100 U/ml), IL-18 (50 ng/ml), and IL-2 (100 U/ml) + IL-18 (50 ng/ml) for 18 h at 2 x 106 cells/ml. The cell-free supernatants were harvested, and cytokines were measured by ELISA. No significant levels of IFN-{gamma} were found in the supernatants from IFN-{gamma} (-/-) mice. The purity of CD3- and DX5- cells isolated from C57BL/6 mice was 95.4% and 97.5%, respectively. The purity of CD3- and DX5- cells isolated from IFN-{gamma} (-/-) mice was 97.9% and 99.0%, respectively. The figures show the data from one representative experiment. The ELISA values have an error range of within 10%.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-13 was originally cloned from a murine Th2 cell clone and initially designated as P600 (4). IL-13 is a pleiotropic cytokine involved in the development of humoral immunity (4). IL-13 shares biological functions with IL-4, but has only 30% of sequence homology with IL-4 (4, 17). Many studies have demonstrated that IL-4 can promote Th2 CD4+ and CD8+ T cell development from naive T cells (4). Although to date there is no evidence that IL-13 can directly drive Th2 cell development, IL-13, like IL-4, is thought to have an important role in the development of the humoral immune response (4). In this study, we have demonstrated that IL-18 strongly coinduces IL-13 mRNA and protein in murine NK and T cells in synergy with IL-2. We have found that in the IFN-{gamma} (-/-) mice, liver and spleen NK and T cells produced more IL-13 mRNA and protein in response to IL-2 + IL-18 than seen in equivalent cells obtained from control B6 mice.

IL-18, originally identified as an IFN-{gamma}-inducing factor, promotes the production of IFN-{gamma} and enhances NK activity (10). Although IL-18 alone cannot induce IFN-{gamma} production by itself, IL-18 synergizes with IL-12 in inducing IFN-{gamma} and GM-CSF production (Th1 cytokines) from T cells and NK cells (9, 10, 11, 12). Despite the fact that IL-18 is not structurally related to IL-12, IL-18 has functional similarities to IL-12 (11, 12). Moreover, it has been reported that IL-18 alone does not drive Th1 development, but strongly potentiates IL-12-driven Th1 development (34). A previous study also showed that IL-18 reduced the expression of the Th2 cytokine, IL-10 (14). Therefore, IL-18 is thought to be an important cofactor involved in Th1 cytokine production and Th1 cell development. However, in this study, we demonstrate that IL-18 induces Th2 cytokine IL-13 production in both NK and T cells in synergy with IL-2 but not with IL-12, although IL-12 + IL-18 or IL-2 + IL-18 synergistically induce IFN-{gamma} production in NK and T cells. Recent studies from our laboratory revealed that in the human NK3.3 cell line, IL-13 production induced by IL-2 was not modulated by IL-12, although IL-2 and IL-12 synergistically induced IFN-{gamma} production (24). This result is consistent with the data presented in this study. Moreover, in the absence of IFN-{gamma} (i.e., IFN-{gamma} (-/-) mice), IL-2 + IL-18-activated NK and T cells produced more IL-13 mRNA and protein than normal controls. It has been established that IL-18 alone does not drive Th1 nor Th2 development, but can be a strong cofactor for Th1 cell development in combination with IL-2 or IL-12 (11, 12, 34). However, our results suggest that in the absence of IFN-{gamma} (or when the levels of IFN-{gamma} are suppressed), IL-18 may be a cofactor for the development of humoral immunity in synergy with IL-2. The fact that significantly more IL-13 was produced in NK and T cells obtained from IFN-{gamma} (-/-) mice suggests that IFN-{gamma} levels may endogenously regulate IL-13 production by IL-18 and IL-2 in vivo.

It has been reported that IL-12 is an inducer of IL-10, as well as IFN-{gamma} gene expression, in combination with IL-2 in both T and NK cells (7, 22). Our results also show that in IFN-{gamma} (-/-), RAG-2 (-/-), and control mice, IL-10 was induced by IL-12 in synergy with IL-2. IL-18 + IL-12 or IL-18 + IL-2 weakly induced IL-10 production in these mice (Fig. 4Go). These results suggest that IL-18 is not a strong inducer of IL-10. Moreover, IL-10 production induced by IL-12 and/or IL-2 may be IFN-{gamma}-independent, as induction levels were the same in cells obtained from B6 and IFN-{gamma} (-/-) mice.

It is unclear whether IFN-{gamma} directly affects the IL-13-producing cells identified in this report. There are several possibilities that could account for the results that we have presented. Previous studies demonstrated that IFN-{gamma} inhibited the development of Th2 cell clones producing IL-4 (35, 36). Therefore, one possibility is that IFN-{gamma} can suppress the initial development of these IL-13-producing NK and T cells from precursors in the bone marrow, and, in the absence of IFN-{gamma}, these cells predominate. Alternatively, IFN-{gamma} may induce growth factor(s) that directly suppress the development of these IL-13-producing cells. It is also possible that as the precursors of IL-13-producing cells mature, these cells might lack IFN-{gamma} receptors and become refractory to IFN-{gamma} effects. In preliminary experiments, we have observed that in vitro IFN-{gamma} treatment partially blocked IL-13 production by IL-2 in cells obtained from IFN-{gamma} (-/-) mice (data not shown), thus, suggesting a potential direct effect of IFN-{gamma} on IL-13 gene expression. Furthermore, in transient transfection experiments, we found that IFN-{gamma} partially inhibits IL-13 promoter activity (data not shown). Thus, another possibility is that IFN-{gamma} directly inhibits IL-13 production in IL-13-producing cells via IFN-{gamma} receptor signaling. Recent studies have shown that a strong Th2 cell response was observed in an infectious disease model using IFN regulatory factor (IRF)-1 (-/-) mice (37, 38). As IFN-{gamma} directly activates IRF, it is possible that IRF-1 could directly or indirectly alter IL-13 promoter activity. Therefore, it will be of interest to determine whether IL-2 or IL-2 + IL-18 can induce IL-13 expression in IRF-1 (-/-) mice.

It has been reported that IL-4/IL-13-producing Th2 cells in vivo can be a small population of the total conventional T cells (39). Several reports have shown that the NK-T subset (NK1.1+ T cell) can produce more IL-4 than conventional T cell subsets (31, 32, 33). However, it is still unclear which population (conventional T, NK-T, or a NK subset) is the major population producing IL-13. In this study, we have demonstrated that, in IFN-{gamma} (-/-) and B6 mice, liver lymphocytes can produce more IL-13 mRNA and protein than spleen lymphocytes in response to IL-2 + IL-18. FACS analysis revealed that in IFN-{gamma} (-/-) and B6 mice, a larger number of NK and NK-T cells migrated to the liver than to the spleen following in vivo treatment with IL-2. Repeated sorting experiments showed that purified spleen NK cells produced ~3- to 10-fold more IL-13 protein than purified spleen T cells in response to IL-2 + IL-18. Moreover, RPA and ELISA analyses revealed that in IL-2-treated and -untreated RAG-2 (-/-) and SCID mice that lack conventional T, NK-T, and B cells, the levels of IL-13 mRNA and protein production were not significantly different from those observed in cells obtained from control B6 mice following IL-2 + IL-18 stimulation in vitro (Fig. 4Go, data not shown). These results suggest that NK-T and conventional T cell subsets might not be major producers of IL-13 in response to IL-2 + IL-18. It is also worth noting that the liver NK cells isolated from SCID and RAG-2 (-/-) mice produced more IL-13 mRNA and protein than spleen NK cells in response to IL-2 or IL-2 + IL-18 (data not shown). Thus, the NK cells that migrated into the liver and spleen in IL-2-treated mice might represent different NK subpopulations or the expression of the IL-2 receptor or the IL-18 receptor might differ in these populations. Relatively high doses (50 ng/ml) of IL-18 were needed to induce IL-13 production in both B6 and IFN-{gamma} (-/-) mice, while low doses of IL-18 (5 ng/ml) could induce IFN-{gamma} in combination with IL-2 in B6 mice (Table IGo). While the basis for this difference is currently unknown, it may reflect differences in the cell population responding to IL-18. This hypothesis requires further study.

Our present study has demonstrated that, although purified resting spleen T and NK cells did not respond to IL-2 + IL-18, in vivo IL-2-treated spleen cells produce IFN-{gamma} or IL-13 in response to IL-18 in synergy with IL-2 in vitro. Recent studies have revealed that IL-1 receptor-related protein is an IL-18 receptor (40), and that IL-12 can up-regulate the IL-18 receptor in mouse Th1 and B cells (15, 16). These results suggest that resting spleen cells might not express IL-18 receptors, and in vivo IL-2 treatment may up-regulate IL-18 receptor expression. This issue is currently under investigation.

In conclusion, this study has demonstrated that IL-18 is a potent coinducer of the Th2 type cytokine, IL-13, in murine NK and T cells. We have found that in the absence of IFN-{gamma}, liver and spleen NK and T cells produced more IL-13 mRNA and protein in response to IL-2 + IL-18 than seen in equivalent cell populations obtained from control B6 mice. Furthermore, while IL-2 + IL-12 synergized with regard to IL-10 and IFN-{gamma} production, no enhanced IL-13 production was observed. Taken together, our results suggest that when IFN-{gamma} is suppressed, IL-18 can be a cofactor in the development of the humoral immune response by inducing IL-13. Thus, IL-12 and IL-18 have different roles in the regulation of cytokine gene expression that effects the Th1/Th2 balance in response to Ag challenge.


   Acknowledgments
 
We thank Drs. John Ortaldo (Laboratory of Experimental Immunology, Division of Basic Sciences, NCI) and Koji Tamada (Kyushu University, Fukuoka, Japan) for helpful scientific discussions; Dr. Scott Durum (Laboratory of Molecular Immunology, NCI-FCRDC) for providing RAG-2 (-/-) mice; John Wine for animal experiments; Gordon Weigand for cell sorting; Beti Evtimoska and Anna Mason for cytokine analysis; and Joyce Vincent for editorial assistance. Animal care was provided in accordance with the procedures outlined in the "Guide to the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 86-23, 1985).


   Footnotes
 
1 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. Back

2 T.H. is the recipient of a research fellowship from the Uehara Memorial Foundation (Tokyo, Japan) and the 1997 Fukuoka Cancer Society Award (Fukuoka, Japan). Back

3 Address correspondence and reprint requests to Dr. Howard A. Young, Laboratory of Experimental Immunology, National Cancer Institute, Frederick Cancer and Research Development Center, Building 560, Room 31-93, Frederick, MD 21702. E-mail address: Back

4 Abbreviations used in this paper: h, human; RPA, RNase protection assay. Back

Received for publication November 18, 1998. Accepted for publication February 1, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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