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Immune FunctionEvidence of a Broad Role for IL-18 in Modulating Cutting Edge: IL-18-Transgenic Mice: In Vivo

A. Young and Kotaro OizumiKohichiro Yoshino, Ken-ichi Yamamura, Jun-ichi Miyazaki, Howard Tomoaki Hoshino, Yusuke Kawase, Masaki Okamoto, Koichi Yokota,

http://www.jimmunol.org/content/166/12/7014doi: 10.4049/jimmunol.166.12.7014

2001; 166:7014-7018; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/12/7014.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Cutting Edge: IL-18-Transgenic Mice:In Vivo Evidence of a Broad Role forIL-18 in Modulating Immune Function

Tomoaki Hoshino,1* Yusuke Kawase,2†

Masaki Okamoto,2* Koichi Yokota,†

Kohichiro Yoshino,† Ken-ichi Yamamura,‡

Jun-ichi Miyazaki,§ Howard A. Young,¶

and Kotaro Oizumi3*

IL-18 has been shown to be a strong cofactor for Th1 T celldevelopment. However, we previously demonstrated that whenIL-18 was combined with IL-2, there was a synergistic induc-tion of a Th2 cytokine, IL-13, in both T and NK cells. Morerecently, we and other groups have reported that IL-18 canpotentially induce IgE, IgG1, and Th2 cytokine production inmurine experimental models. Here, we report on the genera-tion of IL-18-transgenic (Tg) mice in which mature mouseIL-18 cDNA was expressed. CD81CD44high T cells and mac-rophages were increased, but B cells were decreased in thesemice while serum IgE, IgG1, IL-4, and IFN-g levels were sig-nificantly increased. Splenic T cells in IL-18 Tg mice producedhigher levels of IFN-g, IL-4, IL-5, and IL-13 than control wild-type mice. Thus, aberrant expression of IL-18 in vivo results inthe increased production of both Th1 and Th2 cytokines. TheJournal of Immunology,2001, 166: 7014–7018.

I nterleukin 18 was discovered as IFN-g-inducing factor andacts in synergy with IL-12 to enhance IFN-g gene expression(1–3). IL-18 also induces Fas ligand, GM-CSF, proinflam-

matory cytokines TNF and IL-1b, and chemokines such as IL-8and macrophage-inflammatory protein 1a (2–5). IL-18 shares bi-ological similarity with IL-12, a strong Th1 inducer, althoughIL-18 is not structurally related to IL-12 (1–3). IL-18, like IL-12,augments NK activity through the induction of constitutively ex-

pressed IL-18R on NK cells (6). Moreover, a previous study re-ported that IL-18Ra (IL-1R-related protein) is selectively ex-pressed on the surface of Th1 but not Th2 cells (7). Based on thesereports, IL-18 was thought to be a strong cofactor for Th1 celldevelopment (2, 3, 8). However, we have demonstrated that IL-18,in combination with IL-2 but not with IL-12, can be a strong co-factor for the expression of a Th2 cytokine, IL-13, in T cells andin a unique NK population (9, 10). More recently, we and othergroups have reported that IL-18 can potentially induce Th2 cyto-kines (IL-4, IL-5, IL-10, IL-13) and IgE and IgG1 production (11–16), suggesting that IL-18 can act as a cofactor for both Th1 andTh2 cell development.

IL-18 is intracellularly produced from a biologically inactivatedprecursor; pro-IL-18 and mature IL-18 are secreted after the cleav-age of pro-IL-18 by caspase-1, originally identified as IL-1b-con-verting enzyme (2, 3). It has been reported that pro-IL-18 mRNAis expressed in a wide range of cells, including Kupffer cells, mac-rophages, T cells, B cells, osteoblasts, keratinocytes, dendriticcells, astrocytes, and microglia (2, 3). In fact, pro-IL-18 protein isproduced in various cells including Kupffer cells, macrophages,and keratinocytes, whereas mature IL-18 is only weakly detectedin mouse sera or tissues (1–3). To test the biological consequencesof IL-18 overexpression in vivo, we generated IL-18-transgenic(Tg)4 mice in which B and mature T cells could express maturemouse IL-18 cDNA fused with the signal peptide of the mouse Igk-chain under the control of mouse Ig promoter. The results pre-sented here demonstrate that overexpression of IL-18 can inducehigh IgE, IgG1, IL-4, and IFN-g expression in vivo. Thus, aberrantin vivo IL-18 expression results in the expression of cytokines thataffect both Th1-and Th2-type development, suggesting that theclinical use of this IL may result in unexpected physiologicalconsequences.

Materials and MethodsTransfection

Complementary DNAs encoding the signal peptide (SP) from the V-J2-Cregion of the mouse Igk-chain (17) fused with mature mouse IL-18 cDNA(1) and mouse IL-18 cDNA were generated by PCR using mouse pro-IL-18cDNA (18), kindly obtained from Kiyoshi Takeda (Osaka University,

*Department of Internal Medicine 1, Kurume University, Kurume, Japan;†Researchand Development Division, R&D Laboratories, Nippon Organon K.K., Osaka, Japan;‡Institute of Molecular Embryology Genetics, Kumamoto University, Kumamoto,Japan;§Department of Nutrition and Physiological Chemistry, Osaka UniversityMedical School, Osaka, Japan; and¶Laboratory of Experimental Immunology, Na-tional Cancer Institute-Frederick, Frederick, MD 21702

Received for publication March 5, 2001. Accepted for publication April 17, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Tomoaki Hoshino, Departmentof Internal Medicine 1, Kurume University School of Medicine, 67 Asahi-machi,Kurume 830-0011, Japan. E-mail address: hoshino@med.kurume-u.ac.jp2 Y.K. and M.O. contributed equally to this work.3 Current address: TB Laboratory, University Teaching Hospital, Zambia.

4 Abbreviations used in this paper: Tg, transgenic; SP, signal peptide; m, murine; wt,wild type; EF, elongation factor.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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Osaka, Japan). Amplified DNA was subcloned into the pCR2.1 vector (In-vitrogen, Carlsbad, CA) and sequenced. Then mature mouse IL-18 cDNAencoding SP and mature mouse IL-18 were subcloned into pcdEF3 vector(19), kindly obtained from Jerome Langer (Robert Wood Johnson MedicalSchool, Piscataway, NJ). Plasmid DNA (0.5–2mg) was transfected into3 3 105 293 cells by FuGene 6 (Boehringer Mannheim, Mannheim, Ger-many). The cell supernatants and cell lyses were analyzed using a murine(m) IL-18 ELISA kit (MBL, Nagoya, Japan) and Western blotting, respec-tively, 48 h after the transfection. Rat anti-mIL-18 mAb (39-3F; MBL) orrabbit anti-mIL-18 Ab, kindly obtained from Charles A. Dinarello (Uni-versity of Colorado Health Sciences Center), was used for Westernblotting.

Generation of a Tg mouse expressing mature mouse IL-18

Mature mouse IL-18 cDNA encoding SP (IL-18SP) were generated andsubcloned into the pCR2.1 vector as describe above. TheEcoRI-digestedDNA fragment was inserted into theEcoRI site of the pEmIgH vector (20)containing the human Em enhancer and mouse IgVH promoter. The linearpEmIgH/IL-18SP DNA fragment was injected into fertilized eggs of B6mice at Oriental Bio Service (Kyoto, Japan). Hemizygous Tg mice weregenerated by mating founder mice with wild-type (wt) B6 mice. The off-spring mice were screened by PCR and Southern blotting analyses usingtail DNA as previously reported (21), and IL-18 expression in sera wasconfirmed by the mouse IL-18 ELISA.

Surface Ag and intracellular analysis by flow cytometry

Three-color analysis was performed using a FACScan (22). For intracel-lular cytokine staining (10), isolated spleen cells from Tg and wt mice werestimulated with PMA (20 ng/ml) plus ionomycin (500 ng/ml) for 4 h in thepresence of 4mM monensin at 37°C. Then cells were further stained withFITC-anti-mIL-4, FITC-anti-mIL-5, PE-anti-mIFN-g, and/or control iso-type-matched mAb. A total of.30,000 cells was analyzed in each FACSanalysis.

Statistical analysis

The difference between groups was analyzed by Wilcoxon signed rank testand, if appropriate, by pairedt test. Values ofp , 0.05 were considered tobe significant.

Results and DiscussionExpression of the mature mouse IL-18 transgene in the IL-18 Tgmouse

First, transfection experiments were conducted to confirm whetherthe transgene construct could induce an optimal secretion of ma-ture IL-18 in vitro. Expression from two constructs was analyzed:one was mature mouse IL-18 cDNA fused to the SP of the mouseIg k-chain, and the other was mature mouse IL-18 cDNA withoutthe SP used as a control. These two cDNA constructs were sub-cloned into the pcdEF3 vector containing the human elongationfactor (EF) 1a promoter (19) and were designated as pEF-IL-18SPand pEF-IL-18, respectively. Of these expression constructs, 0.5–2mg was transiently transfected into 293 cells. Mature IL-18 wasfound in the supernatants of the pEF-IL-18SP but not in pEF-IL-18-transfected 293 cells (Fig. 1A). Western blotting analysisshowed both pEF-IL-18SP- and pEF-IL-18-transfected 293 cellsproduced mature IL-18 protein of;18 kDa (data not shown).Based on the expression of soluble IL-18, we generated an IL-18Tg mouse under the control of murine Ig promoter and human Igenhancer. The construct design is shown in Fig. 1B. We estab-lished 5 founders (no. 8 strain male, no. 13 male, no. 14 male, no.23 female, and no. 24 female) that showed transgene integration.For most studies shown below, we used hemizygous mice from theno. 8 founder. ELISA analysis showed high serum IL-18 levelswere found in all IL-18 Tg founders and hemizygous mice derivedfrom these founders, whereas mean serum IL-18 levels of controlwt B6 mice were,0.26 ng/ml. Large amounts of mature IL-18 (18kDa) protein were detected in the sera of IL-18 Tg mouse by West-ern blot analysis (Fig. 1C).

The IL-18 Tg mouse has increased numbers of CD81 T cellsand macrophages, but decreased numbers of B cells in thespleen

Ten- to 14-wk-old Tg and control mice (n 5 4 each) wereanalyzed by flow cytometry, and a representative staining pat-tern is shown in Fig. 2A. We found that the mean total numberof cells in the Tg and wt mice was 1.03 108 and 1.23 108

cells/spleen, respectively. The mean percentage of CD81CD42

but not CD41CD82 T cells was significantly (p , 0.05) in-creased in the Tg mice when compared with wt mice(CD81CD42 T cells in Tg 23.3% vs wt 12.7%). Spleen CD81

T cells in the Tg mice showed a high CD44 (CD44high) CD25(IL-2Ra2) phenotype, whereas;25% of CD41 T cells in theTg mice expressed CD25 (data not shown). In contrast, thepercentage of CD191B2201IgM1 B cells was significantly( p , 0.05) decreased when compared with wt mice (Tg 32.4%vs wt 54.4%). The percentages of CD32 NK1.11 DX51 NKcells, CD31 NK1.11 DX51 (NK-T), and CD31 TCRgd1 cellsin Tg mice were;2– 4%, and no significant difference wasfound when compared with wt mice. CD3 and TCRab expres-sion on T cells in the Tg mice was weaker than that observed inwt mice (data not shown). Moreover, the Gr11 CD11b(Mac1a)1 F4/801 population was increased in the Tg mice.

FIGURE 1. Generation of the IL-18 Tg mouse.A, 0.5–2mg of constructbearing mature IL-18 cDNA fused with the SP from the mouse Igk-chain(IL-18(SP)) or mature IL-18 cDNA without the SP (IL-18) under the con-trol of the human EF-1a promoter (pcdEF3 vector (19)) was transientlytransfected into 33 105 293 cells utilizing FuGene 6 (Boehringer Mann-heim). The supernatants were analyzed 48 h after the transfection using amIL-18 ELISA kit. ND, not detectable.B, pEmIgH vector encoding humanEm enhancer and mouse IgVH promoter (20) was used to generate IL-18Tg mice. A schematic design of the cDNA construct used to generate theIL-18 Tg mouse is shown.C, Serum IL-18 levels in IL-18 Tg (no. 8 strain)and littermate wt C57BL/6 mice (n5 4 each) were measured using amIL-18 ELISA kit (MBL). Western blotting analysis for detecting serummature IL-18 (18 kDa) in the IL-18 Tg and wt mice was performed.

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These results indicate that memory-phenotype CD81CD44high

T cell and Gr11 CD11b (Mac1a)1 F4/801 macrophage or gran-ulocyte populations were selectively expanded in the spleen ofIL-18 Tg mice.

CD41 T cells express intracellular IL-4, IL-5, and IFN-g in anIL-18 Tg mouse

Naive spleen cells isolated from Tg and wt mice were stimulatedwith PMA plus ionomycin for 4 h and analyzed for cytoplasmicIL-4, IL-5, and IFN-g expression in CD41 and CD81 T cells (Fig.2B). The intracellular staining revealed that CD41 T cells in Tgmice strongly expressed IFN-g and the type 2 cytokine IL-5, butweakly expressed IL-4. Although CD81 T cells barely expressedIL-4 in both Tg and wt mice, CD81 T cells in Tg mice stronglyexpressed IFN-g and IL-5. We could not analyze IL-13 expressionon CD41 and CD81 T cells, as there is no commercial reagent forintracellular staining of mIL-13.

IL-4, IL-5, IL-13, and IFN-g production in vitro culture in anIL-18 Tg mouse

As we have previously reported (9–11), naive spleen lymphocytesfrom wt mice did not induce the Th2 cytokines, IL-4, IL-5, and

IL-13 (,40 pg/ml), but induced IFN-g production in response toPMA plus ionomycin, anti-CD3 mAb, IL-2 alone, or anti-CD3mAb plus IL-2 (Table I). Surprisingly, naive spleen lymphocytesfrom Tg mice did demonstrate greater production of IFN-g, IL-5,and IL-13 when compared with wt mice in response to these stim-uli. In contrast, IL-4 production was not significantly increasedwhen supernatants from the Tg mouse spleen cells and controlcells were compared. The same observation was found in the invitro culture using nylon wool column-passed spleen lymphocyteswhere equivalent numbers of CD31 T cells are present in both Tgand wt mice (data not shown). Our present study supports previousreports which demonstrate that IL-13 and IL-5 may be more af-fected by IL-18 than IL-4 (11–13).

Serum levels of IgE, IgG1, IL-4, and IFN-g were increased inthe IL-18 Tg mouse

We analyzed serum IgE, IgG1, IL-4, and IFN-g levels in 9-wk-oldTg and wt mice (Fig. 3). Serum IgE levels of Tg mice were sig-nificantly (p 5 0.01) higher than those observed in control wt B6mice (,0.4mg/ml), whereas serum IgG1 levels were not signifi-cantly increased. Surprisingly, serum IgG1 levels in Tg mice grad-ually increased with aging. Serum IgG1 levels in 15-wk-old Tg

FIGURE 2. CD81CD44high T cells and macrophages were increased, but B cells were decreased in the IL-18 Tg mouse.A, Spleen cells were isolatedfrom IL-18 Tg and littermate wt mice and stained with FITC-, PE-, or CyChrome-conjugated mAb in the presence of anti-mCD16/CD32 mAb.B, SpleenCD41 T cells can express IL-4, IL-5, and IFN-g in the IL-18 Tg mouse. Isolated spleen cells were cultured with PMA (20 ng/ml) plus ionomycin (500ng/ml) for 4 h, washed, and stained with CyChrome-conjugated anti-mCD4 or anti-mCD8 mAb in the presence of anti-mCD16/CD32 mAb. Then cells werefixed, permeabilized, and stained with PE-anti-mIFN-g mAb and FITC-anti-mIL-4 mAb or FITC-anti-mIL-5 (PharMingen, San Diego, CA). Three-coloranalysis was performed for analyzing cytoplasmic IL-4, IL-5, and IFN-g expression in CD41 and CD81 T cells.

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mice were significantly (p 5 0.02; ;3-fold) higher than thoseobserved in 9-wk-old Tg mice and were also significantly (p 50.02) higher than in 15-wk-old wt mice. Normally, serum IL-4 andIFN-g levels were below the detectable level. Serum IL-4 levelswere significantly (p 5 0.04) increased in Tg mice, although IL-4expression and production in the spleen cells of IL-18 Tg mice wasweak (Table I and Fig. 2B). Yoshimoto et al. (12) previously re-ported that IL-18 induced IL-4 production by basophils. Theseresults suggest that other lymphoid organs can produce IL-4 in Tgmice. Serum IFN-g levels in Tg mice were significantly (p 50.04) increased compared with control wt mice, consistent withthose observed in intracellular staining and in vitro analyses. It isalso possible that increased IgE and IgG1 levels and cytokineslevels were influenced by the fact that the IL-18 Tg mice containmore T cells and are more endogenously activated in vivo. Furtheranalysis is needed to test this hypothesis.

Although it is known that IL-4 and IL-13 are critical for Th2development, other factors that can up-regulate Th2 cytokines (8),including IL-4, IL-5, IL-10, and IL-13 and IgE production, arepoorly defined. A previous paper has demonstrated that IL-1a, butnot IL-18 can amplify Th2 but not the Th1 response, and thatIL-1a and IL-18 may differentially induce Th1 and Th2 responses,respectively (23). In the present study, however, overproduction ofIgE, IgG1, Th2 cytokines, IFN-g, but not IL-1a, and IL-1b wasobserved (data not shown). Thus, both Th1- and Th2-like re-sponses were initiated by the presence of the IL-18 transgene invivo and in vitro. These results suggest that although a defectiveTh1 response was reported in IL-18-deficient (2/2) mice (18),overexpression of IL-18 can result in expression of cytokines thatcan drive both Th1 and Th2 development in an IL-1-independentfashion. Further studies, using IL-12/2 mice, will be needed to testthe role of IL-1 in this model.

CD81 T cells can be classified into two distinct effector celltypes based on their cytokine-secreting profiles following Ag stim-ulation. Type 1 CD81 T cells (Tc1) produce IL-2, IFN-g, andTNF-a, whereas type 2 CD81 T cells (Tc2) predominantly expressIL-4, IL-5, and IL-10 (8). We observed increased numbers ofCD81CD31 T cells in the spleen of IL-18 Tg mice. Intracellularstaining analysis revealed that CD81 T cells in Tg mice stronglyexpressed IFN-g and IL-5, but barely expressed IL-4 when stim-ulated with PMA plus ionomycin (Fig. 2B). These results suggestthat the increased CD81 T cells in Tg mice are neither Tc1 nor Tc2like as they express both IFN-g and IL-5.

Perturbation of T cell and thymocyte development was previ-ously reported in IL-4 and IL-13 Tg mice (24). Previously, weestablished IFN-g Tg mice in which bone marrow and thymocyteswere expressing IFN-g, resulting in the absence of all B cells, Tcell lineage alterations, and hemopoietic progenitor deficiencies

(21). In the IL-18 Tg mice, while CD81CD44high T cells and F4/801 macrophages were induced, B cells were reduced, when com-pared with wt mice. Thus, we hypothesize that the changes inlymphocyte populations in the IL-18 Tg mice are due to in vivoinduction of IFN-g, IL-4, IL-13, and other cytokines or chemo-kines. Cross-breeding these mice with mice lacking specific cyto-kine genes will be necessary to test this model.

In summary, our data demonstrate that the expression of IL-18in vivo can modulate IgE, IgG1, IFN-g, and Th2 cytokines and Tcell, B cell, and macrophage development. IL-18 Tg animals rep-resent an important tool for defining the in vivo and in vitro reg-ulation of Th1 and Th2 development and demonstrate the impor-tance of analyzing the effects of cytokine expression in murinemodels before proceeding to human clinical trials.

FIGURE 3. Serum IgE, IgG1, IL-4, and IFN-g levels were increased inthe IL-18 Tg mouse.A, Serum IgE, IL-4, and IFN-g levels in 9-wk-oldIL-18 Tg and littermate wt mice (n5 4 each) were measured by ELISA aspreviously reported (11). Serum IgE, IL-4, and IFN-g levels in wt micewere,0.4 mg/ml, ,20 pg/ml, and 20 pg/ml, respectively.B, Serum IgG1levels in IL-18 Tg mice gradually increase with aging. Serum IgG1 levelsin IL-18 Tg and littermate wt mice (n5 3 each) were measured in mice 9to 15 wk old. Serum IgG1 levels in 15-wk-old IL-18 Tg mice were sig-nificantly (p 5 0.02) higher in 9-wk-old IL-18 Tg mice and were alsosignificantly (p5 0.02) higher than in 15-wk-old wt control mice.

Table I. Spleen cells in the IL-18-Tg mouse potentially induce both Th1 and Th2 cytokinesa

IFN-g (pg/ml) IL-13 (pg/ml) IL-4 (pg/ml) IL-5 (pg/ml)

Tg wt Tg wt Tg wt Tg wt

None 20a ,10 30a ,8 ,30 ,30 ,20 ,20PMA (20 ng/ml)/ionomycin (500 ng/ml) 52,000b 5,260 1,670b 30 50 40 150b ,20Anti-CD3 mAb (5 mg/ml) 3,320c 360 120c ,8 ,30 ,30 60c ,20IL-2 (1,000 IU/ml) 110d ,10 70d ,8 ,30 ,30 ,20 ,20Anti-CD3 mAb (5 mg/ml) 1 IL-2 (1,000 IU/ml) 8,370e 1,100 280e 30 ,30 ,30 90e ,20

a Spleen cells were isolated from an IL-18 Tg and a littermate wt mouse and stimulated for 18 h at 23 106 cells/ml. The cell-free supernatants were harvested, and cytokineswere analyzed using ELISA kits. ELISA values have an error range within 10%.p , 0.05 vs wt mouse without stimulation.

b p , 0.05 vs wt mouse stimulated with PMA plus ionomycin.c p , 0.05 vs wt mouse stimulated with anti-CD3 mAb alone.d p , 0.05 vs wt mouse stimulated with IL-2 alone.e p , 0.05 vs wt mouse stimulated with anti-CD3 mAb plus IL-2.

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