IL-18 induction of IgE: dependence on CD4 + T cells, IL-4 and STAT6

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1 ARTICLES IL-18 induction of IgE: dependence on CD4 + T cells, IL-4 nd STAT6 Tomohiro Yoshimoto 1,2,7, Hitoshi Mizutni 3, Hiroko Tsutsui 1, Nncy Noen-Truth 6, Kei-ichi Ymnk 3, Minoru Tnk 4, Shinzo Izumi 5, Hruki Okmur 2,Willim E. Pul 6 nd Kenji Nknishi 1,2,7 Overproduction of immunogloulin E (IgE) nd T helper cell type 2 (T H 2) cytokines, including interleukin 4 (IL-4), IL-5 nd IL-13, cn result in llergic disorders. Although it is known tht IL-4 is criticl to the polriztion of nïve CD4 + T cells to T H 2 phenotype, oth in vitro nd in mny in vivo systems, other fctors tht regulte in vivo IL-4 production nd T H 2 commitment re poorly understood. IL-18, n IL-1 like cytokine tht requires clevge with cspse-1 to ecome ctive, ws found to increse IgE production in CD4 + T cells, IL-4 nd STAT6 dependent fshion. IL-18 nd T cell receptor medited stimultion could induce nïve CD4 + T cells to develop into IL-4 producing cells in vitro.thus, cspse-1 nd IL-18 my e criticl in regultion of IgE production in vivo, providing potentil therpeutic trget for llergic disorders. Interleukin 4 (IL-4) is the most importnt determinnt of immunogloulin (Ig) clss switching to IgE 1,2. IL-13 my lso induce clss switching to IgE in humn B cells 3,4. In ddition to regulting IgE production, oth cytokines directly elicit llergic inflmmtory responses such s mucus production y ronchiolr endothelil cells 5,6. IL-4 nd IL-13 oth ind to the α chin of the IL-4 receptor (IL-4R) 7 nd therefore the fctors tht induce IL-4 or IL-13 production re intimtely ssocited with the pthogenesis of llergic disorders. IL-18, originlly designted s interferon (IFN)-γ inducing fctor (IGIF), is pleiotropic cytokine secreted y ctivted mcrophges nd Kupffer cells Its mjor ction is induction of IFN-γ y T H1 cells nd NK cells, especilly in comintion with IL Along with IL-12, IL-18 lso induces nti-cd40 ctivted B cells to produce IFN-γ, which inhiits IL-4 dependent IgE nd IgG1 production 17. Administrtion of IL-12 nd IL-18 in helminth-infected mice inhiits oth IgE production nd production of IL-4 nd IL-13 y sophils nd mst cells in n IFN-γ dependent mnner, thus offering different pproch for the tretment of llergic disorders 17,18. However, it hs lso een reported tht IL-18 enhnces eosinophil recruitment into the irwys 19 nd, in comintion with IL-2, increses IL-13 secretion y NK cells nd T cells 20. We hve shown tht in the presence of IL-3, IL-18 cuses sophils nd mst cells to express high levels of IL-4, IL-13 nd histmine 18. Although in comintion IL-18 nd IL-12 suppress IgE production in helminth-infected mice, injection of IL-18 lone into such mice ctully increses IgE levels nd enhnces production of IL-4 nd IL-13 y sophils, mst nd CD4 + T cells 18. These results tken together suggest tht IL-18 my induce IL- 4 producing CD4 + T cells or condition cells to mke IL-4 in response to ntigenic stimultion. Here we demonstrte tht, when dministered to mice lone, IL-18 induces high IgE expression y B cells. Expression is dependent on CD4 + T cells, IL-4 nd STAT6. Cspse-1 trnsgenic mice 21, with elevted levels of IL-18 in their ser, disply high IgE in their serum which is entirely dependent on STAT6. In ddition, when cultured in dishes coted with nti-cd3 nd nti-cd28, nïve CD4 + T cells stimulted with IL-18 develop into cells tht produce IL-4 in response to in vitro T cell receptor (TCR) enggement. Finlly, we show significntly incresed serum IL-18 levels in ptients with lepromtous leprosy ut not those with tuerculoid leprosy. Results IgE production in BALB/c mice injected with IL-18 To exmine the cpcity of IL-18 to induce IL-4 dependent IgE production in vivo, we gve dily injections of IL-18 (0.1 5 µg) to wildtype BALB/c mice for 13 dys. IL-18 cused dose-dependent increse in serum IgE, reching 8 µg/ml in response to IL-18 doses of 5 µg/dy n 80-fold increse compred with serum IgE oserved in control mice (Fig. 1). In mice tht received IL-18 doses of 5 µg/dy, oth IL-4 nd IL-13 were detectle in serum. Normlly these cytokines re elow the level of detection ut in the treted mice pek concentrtions of IL-4 (280 pg/ml) nd IL-13 (480 pg/ml) were oserved on dys 8 nd 11, respectively (Fig. 1). Induction of serum IL-4 nd IL-13 ws lso dependent on the IL-18 dose dministered. IL- 18 doses of 1 µg/dy modertely induced these cytokines (40 pg/ml of IL-4 nd 110 pg/ml of IL-13 in ser on dys 8 nd 11, respectively). To identify whether, in response to IL-18, increses in IgE nd in serum IL-4 nd IL-13 were dependent on CD4 + T cells, mice were treted with either nti-cd4 or control ntiody nd injected dily with 5 µg IL-18. Anti-CD4 tretment diminished IgE levels y 90% on dys 1 Deprtment of Immunology nd Medicl Zoology, 2 Lortory of Host Defenses, Institute for Advnced Medicl Sciences, Hyogo College of Medicine, Nishinomiy, Hyogo, , Jpn. 3 Deprtment of Dermtology, 4 Deprtment of Biochemistry, Mie University, Fculty of Medicine,Tsu, Mie , Jpn. 5 Ntionl Leprosrium, Oshim Seisho-En, Kgw , Jpn. 6 Lortory of Immunology, Ntionl Institute of Allergy nd Infectious Diseses, Ntionl Institutes of Helth, Bethesd, MD , USA. 7 Core Reserch for Evolutionl Science nd Technology, Jpn Science nd Technology Corportion,Tokyo, Jpn. Correspondence should e ddressed to K.N. (nkken@hyo-med.c.jp). 132 nture immunology volume 1 no 2 ugust

2 ARTICLES c d (Fig. 2), wheres mice treted with sil-13rα2-fc mounted norml IgE response (Fig. 2). This estlished tht IgE responses to IL-18 re dependent on IL-4 ut not on IL-13. We lso exmined responsiveness of STAT6 / mice 25 to IL-18 tretment nd found tht IgE expression ws not induced (dt not shown), which further sustntited these oservtions. Bsed on these results, it seems resonle to conclude tht CD4 + T cells nd IL-4 re essentil for IL-18 medited induction of IgE nd tht the in vivo production of IL-4 nd IL-13 lso depends upon CD4 + T cells. This is consistent with, ut does not fully estlish, the conclusion tht IL-18 tretment results in the ppernce of IL-4 nd IL- 13 producing CD4 + T cells tht re required for IgE production. However, oth IL-4 / nd IL-4Rα / mice showed undiminished increses in serum IL-13 in response to tretment with IL-18 (Fig. 2c) indicting tht the cpcity of CD4 + T cells to express or cuse expression of IL-13 (nd presumly IL-4) is not IL-4 dependent. Figure 1. IL-18 induced IgE, IL-4 nd IL-13 production in vivo is dependent on CD4 + T cells. () BALB/c mice (5 mice per group) were dily injected with PBS uffer or IL-18 for 13 dys.they were led on 14 dy nd serum IgE ws mesured y ELISA. () BALB/c mice (5 mice per group) were injected dily with PBS uffer or IL-18 (5 µg/dy). Serum IL-4 (filled rs) nd IL-13 (open rs) were mesured y ELISA s indicted. Results re geometric mens ± s.d. (c,d) BALB/c mice were injected dily with PBS uffer (open rs in c) or IL-18 (5 µg/dy) for 13 dys nd lso received nti-cd4 (GK1.5; 0.5 mg/dy, five mice per group, shded rs in c) or control ntiody (rt IgG2; 0.5 µg/dy, 5 mice per group, filled rs in c) 4 nd 7 dys efore IL-18 tretment nd 0, 3 nd 7 dys fter tretment. (c) IgE expression in ser ws mesured on indicted dys. (d) IL-4 (filled rs) nd IL-13 (open rs) on dy 11 ws mesured. Results re geometric mens ± s.d. (*, <0.5 ng/ml.) 11 nd 14 nd inhiited the induction of serum IL-4 nd IL-13, wheres control ntiody tretment did not (Fig. 1c,d). This indicted tht incresed expression of oth IgE nd serum IL-4 nd IL-13, in response to IL-18, ws dependent on CD4 + T cells. IgE production in IL-18 injected IL-4Rα / or IL-4 / mice The importnce of the T H2 cytokines IL-4 nd IL-13 in inducing IgE expression ws exmined y treting BALB/c IL-4Rα chin-deficient (IL-4Rα / ) mice 22 with IL-18. These mice filed to produce IgE in response to IL-18 (Fig. 2). As the IL-4Rα chin is required for responses to oth IL-4 nd IL-13 7, we lso exmined IgE production y IL-4-deficient (IL-4 / ) BALB/c mice 23 nd y mice tht hd een treted with solule IL- 13Rα2-IgGFc fusion protein (sil-13rα2-fc), which neutrlizes IL-13 24, in response to IL-18 injection. IL-4 / mice showed no increse in IgE IgE production in cspse-1 trnsgenic mice Next we tested whether endogenously ccumulted IL-18 resulted in high serum IgE. Trnsgenic mice in which cspse-1 is overexpressed in kertinocytes (cspse-1 Tg mice) hve high serum IL-18 (5.2 ± 1.0 ng/ml t 6-weeks-old nd 7.8 ± 0.7 ng/ml t 12-weeks-old, where n = 5) through intrcellulr processing of pro IL-18 y cspse We found these mice hd high serum IgE nd IgG1 t 8 weeks (Fig. 3), which incresed further y 12-weeks-old. This IgE response does not occur in cspse-1 Tg mice lcking STAT6 (Fig. 3), lthough these mice hd high IL-18 (8.0 ± 1.5 ng/ml). As cspse-1 Tg mice lso produce other cytokines (such s IL-1β), we deleted their Il18 gene y crossing with IL-18 / mice 26. The cspse-1 Tg IL-18 / mice (Fig. 3c) hd significntly diminished serum IgE compred to their IL-18 +/ littermtes (Fig. 3c). Residul IgE my e induced y the ction of cytokines other thn IL-18 nd IL-1β ecuse those trnsgenic mice in which IL-1α, the iologicl functions of which re sme s those of IL- 1β, is overexpressed y kertinocytes do not constitutively produce IgE (H. Mizutni, unpulished oservtion). Thus, IL-18 is principlly responsile for inducing IgE in cspse-1 Tg mice, lthough other fctors my e involved in induction of IgE production. CD40L expression chnges on CD4 + T cells We chrcterized CD4 + T cells derived from mice tht hd een injected dily with 5 µg of IL-18 for 13 dys. These cells expressed concentrtions of CD40 lignd (CD40L) mrna comprle to those oserved y CD4 + T cells isolted from mice treted with helminth the Nippostrongylus rsiriensis week erlier (dt not shown). As we could induce IL-4 dependent IgE production y dministrtion of IL-18 lone into BALB/c mice (Fig. 1), we exmined whether c Figure 2. IL-18 induced IgE production in vivo is dependent on IL-4. BALB/c mice nd BALB/c-ckground IL-4Rα / nd IL-4 / mice (5 mice per group) were injected dily with PBS uffer or IL-18 (5 µg/dy) for 13 dys. IL-18 injected BALB/c mice (5 mice per group) either received no further tretment or received dditionl i.p. injections of control humn IgG (1 µg) or sil-13rα2 Fc (1 mg) every two dys. (,) Serum IgE ws mesured on dy 14 nd (c) serum IL-13 on dy 11. Results re geometric mens ± s.d. (*, <10 pg/ml.) ugust 2000 volume 1 no 2 nture immunology 133

3 ARTICLES c Figure 3. IL-18 secreted in cspse-1 Tg mice induced IgE production in vivo y ctivtion of STAT6. () Serum immunogloulins in cspse-1 Tg mice (n = 3) were mesured t 8-weeks-old.We estlished the cspse-1 Tg STAT6 / mice () nd cspse-1 Tg IL-18 / mice (c) y crossing the knock-out mice with cspse-1 Tg mice. Serum IgE in mice ws mesured t 8-week-old. (*, <0.5ng/ml.) (In, +/ is heterozygous for STAT6; / is homozygous for STAT6. In c, +/ is heterozygous for IL-18; +/ is homozygous for IL-18. IL-18 cn ct directly on nïve T cells to induce the cpcity to express IL-4 nd to help in IgE induction. As source of nïve CD4 + T cells, we used CD4 + T cells derived from ovlumin (OVA)-specific TCR trnsgenic BALB/c mice (DO11.10) 27. We treted them with IL-18 in the sence of OVA for 4 dys in vitro. CD40L ws modestly induced on these cells; induction ws enhnced y the ddition of IL-2 (Fig. 4). Addition of even low doses of IL-18 (5 ng/ml) long with IL-2 induced sustntil increse in CD40L-positive T cells (16.4%). CD4 + T cells stimulted using higher doses IL-18 (50 ng/ml) nd IL-2 (200 pm) expressed detectle ut reltively modest mounts of IL-4 (380 pg/10 6 cells/ml; dt not shown) nd were le to induce B cells to secrete IgE (260 ng/ml) if IFN-γ were neutrlized (Fig. 4). Nïve CD8 + T cells filed to express IL-4 or IL-13 in response to IL-2 nd IL- 18 (dt not shown). These results leve some questions unnswered. Does IL-18 tretment of norml mice induce IL-4 nd IL-13 production nd IgE synthesis y cusing CD4 + T cells to ecome T H2 cells tht help B cells to switch, oth y expressing CD40L nd producing IL-4? Or does this tretment enhnce the cpcity of sophils (or some other cell type) to produce IL-4 in response to T cell dependent IL-3 production? Indeed we hve lredy reported tht mice treted with IL-18 develop popultions of sophils with heightened cpcity to produce IL The optiml stimulnt of IL-4 production y these cells ws IL-3 plus IL-18. However, n enigm remins no ovious stimulnt for IL-3 production y the CD4 + T cells hs een discovered. IL-18 cuses IL-4 dependent in vitro T H 2 polriztion We exmined the effect of IL-18 on T H2 polriztion in vitro (Fig. 5). CD4 + T cells were cultured in nti-cd3 nd nti-cd28 coted dishes for 4 dys with IL-2 lone, IL-2 plus IL-4, or with IL-2 plus IL-18 nd/or nti-il-4. The cells were chllenged with phorol 12-myristte 13-cette (PMA) nd ionomycin nd nlyzed y fluorescence-ctivted cell sorting (FACS) for cytosolic IL-4 nd IFN-γ. T cells cultured with IL-2 nd IL-4 were positive for cytoplsmic IL-4 (11.6%) nd IFN-γ (4.2%). When cultured with IL-2 lone, 3.5% T cells expressed cytoplsmic IL-4 nd 7.1% IFN-γ. However, cytoplsmic IL-4 nd IL- 4 priming ws inhiited y nti IL-4 indicting tht it depended on in situ IL-4 production. Culturing nïve CD4 + T cells with IL-2 nd IL- 18 (2 ng/ml) for 4 dys led to n increse in cytoplsmic IL-4 positive cells (to 10.4%) ut decrese in cytoplsmic IFN-γ positive cells from 7.1% to 3.1%. Higher concentrtions of IL-18 (20 ng/ml) modestly incresed the proportion of cytoplsmic IL-4-positive cells (to 12.1%), ut this could e completely locked y the ddition of nti IL-4 (to 0.4%). Simultneously, we exmined the cpcity of these stimulted T cells to produce IL-4 nd IFN-γ in response to nti-cd3 nd nti-cd28 in vitro chllenge (Fig. 5). Like T cells primed with IL- 4, T cells cultured with IL-18 produced IL-4, which ws completely inhiited y ddition of nti IL-4. This tretment disclosed the originl cpcity of IL-18 to ct on T H1 cells to increse IFN-γ production. Thus, IL-18 induction of T cell cpcity to produce IL-4 is IL- 4 dependent. IL-18 my either increse the mount of IL-4 produced Figure 4. IL-18 stimulted CD4 + T cells to up-regulte CD40L expression nd to cuse B cells to produce IgE in vitro. () Nïve CD4 + T cells ( /0.2 ml/well) from DO11.10 trnsgenic mice were cultured with vrious concentrtions of IL-18 (0 500 ng/ml) in the presence or sence of IL-2 (200 pm).after 4 dys of culture, surfce expression of CD40L ws nlyzed y flow cytometry.the percentges shown represent the proportion of CD40L positive cells mong CD4 + T cells. () Nïve CD4 + T cells ( /0.2 ml/well) from DO11.10 trnsgenic mice were cultured with medium lone, or IL-18 (50 ng/ml) nd/or IL-2 (200 pm).after 4 dys of culture freshly purified BALB/c splenic B cells ( /0.2 ml/well) were dded with or without nti-ifn-γ (10 µg/ml). After n dditionl 10 dys of incution, superntnts were collected nd IgE contents mesured y ELISA. Results re geometric mens ± s.e.m. 134 nture immunology volume 1 no 2 ugust

4 ARTICLES Figure 5. IL-18 stimultes nïve CD4 + T cells to develop into T H2 cells in the presence of TCR enggement in vitro. Nïve CD4 + T cells ( ) from C57BL/6 mice were cultured with 200 pm of IL- 2 plus immoilized nti- CD3 nd nti-cd28 (ech 5 µg/ml) in the presence of IL- 4 (1000 U/ml) or IL-18 (0, 2 nd 20 ng/ml) with or without nti IL-4 (11B11; 10 µg/ml) in 24-well plte in totl of 1 ml.after initil priming for 4 dys, cells were wshed nd recultured with PMA (50 ng/ml) plus ionomycin (500 ng/ml) for 4 h nd nlyzed y FACS for () cytosolic IL-4 nd IFN-γ or () recultured with immoilized nti-cd3 nd nti-cd28 (ech 5 µg/ml for coting) for 24 h, for induction of cytokine secretion. erly in the culture, which cts with nti-cd3 nd nti-cd28 to induce T H2 differentition, or it my increse the sensitivity of these cells to IL-4. High serum IL-18 in lepromtous leprosy Finlly we investigted IL-18 dependent T H2 responses in nturlly occurring humn infectious diseses cused y Mycocterium lepre. There re two polr forms of the disese: tuerculoid leprosy (TL) nd lepromtous leprosy (LL). T H1-devited (in TL) nd T H2-devited (in LL) immune responses to intrcellulr microes ply criticl role in determintion of the lesions 28, lthough mny ptients elong to less cler, intermedite group (IG). We mesured serum IL-12 nd IL-18 in ptients with LL efore (n = 33) or fter (n = 16) tretment with ntileprosy drugs. We lso tested serum IL-18 in ptients with TL (n = 6) or IG (n = 6). As shown in Fig. 6, serum IL-18 ws significntly incresed in ptients with LL, ut ws lmost equivlent etween ptients with TL nd helthy individuls. The IG hd intermedite serum IL-18. Although we mesured serum IL-12 simultneously, it ws undetectle or remined within norml rnge (dt not shown). Effective tretment cused significnt reduction in serum IL-18, suggesting tht expression of IL-18 cn lso e used s good prmeter for mesuring ctivity of the disese. These results indicted tht IL-18 my e involved in T H2 immune responses to M. lepre. Discussion Here we show tht elevtion of IL-18, either y dministrtion or s result of endogenous production, resulted in incresed serum IgE. Production of IgE ws STAT6-dependent nd, t lest in the cse of IL- 18 dministrtion, IL-4 dependent. In ddition, injected IL-18 resulted in the ppernce of serum IL-4 nd IL-13. The increse in IL-13 (nd presumly IL-4) ws independent of IL-4 indicting tht IL-18 Figure 6. IL-18 expression in ptients suffering from leprosy. Serum IL-18 in LL ptients efore (n = 33) or fter (n = 16) tretment with nti-leprosy drugs, ptients suffering from TL (n = 6) or elonging to the IG (n = 6) were mesured. Results re geometric mens ± s.d. **, <0.0005; *; not significnt. IL-18 levels in the IG re not significntly different from those in the other groups. Horizontl line indictes level of IL-18 in the ser of helthy volunteers (n = 20). provides potentil mechnism for generting the IL-4 needed to induce T H2 responses. IL-18 is produced y mny cell types, most notly Kupffer cells 9 nd kertinocytes 29 ; stimulnts such s lipopolyscchride (LPS) induce its secretion, possily y the induction of enzymes cple of cleving pro IL-18 9,30. Induced IL-18 production provides potentil mechnism through which the innte immune response cn moilize IL-4 nd IL-13 production. The nture of cells tht produce IL-4 in response to IL-18 is still uncler, lthough IL-4 nd IL-13 production is T cell dependent. Indeed the simplest interprettion of these results is tht IL-4 is produced y CD4 + T cells ecuse tretment with nti-cd4 prevents the ppernce of serum IL-4 nd IL-13. However, it is known tht IL-3 produced y CD4 + T cells cn induce or enhnce the production of IL-4 y sophils nd mst cells 31, so tht the role of CD4 + T cells in IL-4 production could e indirect. The oservtion tht dministrtion of IL-18 resulted in detectle serum IL-4 nd IL-13 is provoctive. Whtever the cellulr source of IL-4 produced in response to IL-18, it ppers to e induced in sufficient mounts tht concentrtions of IL-4 in the extrcellulr fluid my e dequte to polrize CD4 + T cells responding to n immunologic stimulus to T H2 phenotype. This is importnt s it hs generlly een elieved tht IL-4 cts loclly nd tht the IL-4 producing cells importnt in T H2 polriztion need to e in the immedite vicinity of the developing T cells. This view hs implicted T cells themselves s eing the most importnt source of such IL-4. We hve demonstrted tht IL-18, either dministered or ccumulted, could induce IgE production in vivo without ntigenic stimultion. However, for induction of in vivo IgE production we hd to dminister reltively high doses of IL-18 ( 5 µg/dy per mouse). Moreover, the cpcity of CD4 + T cells, which hd een stimulted with IL-18 (50 ng/ml) nd IL-2 (200 pm) for 4 dys, to induce clss switching to IgE in B cells ws modest. These results my rise the question of pthophysiologicl role for IL-18 in the IgE response. To ddress this issue we exmined the cpcity of IL-18 to promote T H2 polriztion in vitro nd showed tht reltively low concentrtions of IL-18 (2 ng/ml) strongly promoted in vitro T H2 development. As IL- 18 filed to induce T H2 development in the presence of nti IL-4, there re two possile conclusions. IL-18 my increse the erly production of IL-4 nd in comintion with nti-cd3 nd nti-cd28, cuses nïve T cells to develop into T H2 cells. Or IL-18 enhnces the sensitivity of cells to fixed mount of IL-4. Thus, IL-18 with IL-12 selectively ugust 2000 volume 1 no 2 nture immunology 135

5 ARTICLES stimultes T H1 cells to produce IFN-γ nd IL-2 13,14, ut IL-18 y itself induces T H2 response. These results imply tht n infectious gent or n llergenic chllenge tht cuses IL-18 secretion without IL-12 production my induce sufficiently roust nd generlized IL-4 production so tht developing T cells, even if they re not in the immedite vicinity of the IL-4 producing cells, cn receive sufficiently strong IL-4 stimulus to develop into T H2 cells. As we originlly discovered IL-18 in the ser of mice sequentilly treted with Propionicterium cnes nd LPS 8-11, IL-18 production hs een intimtely ssocited with IL-12 production, leding to synergistic induction of IFN-γ production However, we demonstrted tht ptients with lepromtous leprosy T H2-polrized disese 28 hve significntly higher serum IL-18 thn ptients with tuerculoid leprosy T H1-polrized disese 28. Thus, IL-18 my e n importnt determinnt in the development of T H2 cells, lesions nd the clinicl course of M. Lepre infections. At the very lest IL-18 expression correltes well to the ctivity of leprosy. Although this disese is not n exmple of diseses cused y IgE-medited immedite type hypersensitivity, our oservtions suggest tht there re some circumstnces in which IL-18 is secreted independently of IL-12. Our results indicte the potentil involvement of IL-18 (without IL- 12) in the pthogenesis of llergic disorders. It is striking tht IL-18 induced IL-4 nd IL-13 production nd increses in serum IgE without the need for overt immuniztion. Thus it my e tht y inducing vigorous polyclonl IL-4 response, IL-18 production t criticl times in the life of n individul (such s in the neontl period) my therefter contriute to the skewing of the immune response to llergic types of inflmmtory responses. This could e one of the cuses of n topic stte. IL-18 my lso ply n importnt role in the effector lim of llergic responses. Thus, oth irwy epithelil cells 32 nd kertinocytes 29 importnt trgets in sthm nd topic dermtitis, respectively constitutively express IL-18. Stimultion of IL-18 processing enzymes such s cspse-1 could cuse the relese of IL-18. In turn, this could result in production of IL-4 nd IL-13 in the irwys nd the skin in n IgE-independent mnner, where they my directly induce llergic inflmmtory responses. These findings suggest tht cspse-1 nd IL- 18 my e potentil trgets in the effort to develop gents tht regulte llergic inflmmtory responses nd provide new insight into the potentil mechnisms through which T H2 responses my ecome dominnt in n individul. Methods Animls. Specific pthogen-free (SPF) 8-week-old femle C57BL/6 nd BALB/c mice nd BALB/c IL-4 / nd IL-4Rα / mice generted s descried 22,23 nd red under SPF conditions in Ntionl Institute of Allergy nd Infectious Diseses Animl Cre Unit (Rockville, MD) were used. Genertion of STAT6 / or IL-18 / mice ws detiled previously 25,26. Mice trnsgenic for n αβ TCR recognizing OVA( ) (DO11.10; BALB/c genetic ckground) 27 were provided y D. Loh (Wshington University, MO). Kertinocyte-specific humn cspse-1 trnsgenic mice were estlished 21. Offspring were screened for the incorportion of the trnsgene y polymerse chin rection (PCR) nlysis nd Southern lot nlysis using DNA from the til skin. All experiments were performed on the heterozygous trnsgenic line. All mice were red under SPF conditions t the niml fcilities of Hyogo College of Medicine (Nishinomiy, Jpn) nd were used when ged 6- to 10-weeks-old. All niml experiments were conducted ccording to the Guideline for Animl Experiments, Hyogo College of Medicine. In vivo tretment of mice. BALB/c norml, BALB/c IL-4 / or IL-4Rα / mice were injected on dily sis with PBS uffer or IL-18 (0.1 5 µg/dy) for 13 dys. They were led 0, 4, 8, 11 nd 14 dys lter nd serum IgE, IL-4 nd IL-13 were mesured y enzyme-linked immunosorent ssy (ELISA) s descried 18. To deplete CD4 + T cells, BALB/c mice were injected with monoclonl ntiody to CD4 y the intrperitonel (i.p.) route (clone, GK1.5; 0.5 mg/dy) (ATCC: TIB-207) or control ntiody (rt IgG2; 0.5 µg/dy) (Phrmingen) on 7 nd 4 dys efore nd 0, 3, 7 dys fter IL-18 injection. For the in vivo lockde of IL- 13, 1 mg of sil-13rα2-fc or 1 mg of control humn IgG were dministered y the i.p. route every two dys. Estlishment nd purifiction of sil-13rα2-fc ws s descried 24. In vitro ID50, s determined y ility to neutrlize 3 ng/ml of murine IL-13 in the B9 prolifertion ssy, ws pproximtely 10 ng/ml 24. In vitro culture. Splenic CD4 + T cells were purified y MicroBeds (Miltenyi Biotec, Auurn CA). Nïve CD4 + T cells ( /0.2 ml per well) from DO11.10 trnsgenic mice were cultured with vrious doses of IL-18 (0 500 ng/ml) in the presence or sence of IL- 2 (200 pm) for 4 dys in RPMI 1640 supplemented with 10% fetl ovine serum, β mercpto-ethnol (2-ME) (50 µm), l-glutmine (2 mm), penicillin (100 U/ml) nd streptomycin (100 µg/ml). Superntnts were collected nd tested for IL-4 nd/or IL-13 contents y ELISA. The collected T cells were lso exmined for their cpcity to induce B cell to express IgE y incution with the highly purified B cells 17 for n dditionl 10 dys in the presence or sence of 10 µg/ml of ntiody to IFN-γ (R4-6A2, rt IgG1. ATCC: HB-170). Induction of TH1 or TH2 cells. TH1 nd TH2 cells were induced y stimultion of nïve splenic CD4 + T cells ( ) from C57BL/6 mice with 200 pm of IL-2 plus immoilized nti-cd3 nd nti-cd28 (ech 5 µg/ml for coting) in the presence of IL-4 (1000 U/ml) or IL-18 (0, 2 nd 20 ng/ml) with or without nti-il-4 (11B11 33 ); 10 µg/ml) in 24-well plte in totl 1 ml 14,34. After 4 dys of priming, cells (10 5 /0.2 ml per well) were chllenged with immoilized nti-cd3 nd nti-cd28 for 24 h. Superntnts were exmined for IL-4 nd IFN-γ content y ELISA. For intrcellulr cytokine stining, fter priming cells ( /ml) were restimulted with PMA (50 ng/ml) plus ionomycin (500 ng/ml) for 4 h with pulse of 1 µg/ml of Brefeldin A (Phrmingen) during the finl 2 h to inhiit cytokine secretion. Cells were wshed, fixed with 4% (w/v) prformldehyde in PBS uffer, nd the cell memrne permeted with ice-cold PBS contining 1% FCS + 0.1% sponin. Resultnt cells were further stined with phycoerythrin (PE)-rt nti-mouse IL-4 (0.5 µg) plus fluorescein isothiocynte (FITC)-rt nti-mouse IFN-γ (0.5 µg) ntiodies or PE-rt IgG1 plus FITC-rt IgG1 isotype-mtched ntiodies (Phrmingen) for 30 min. Smples were nlyzed on FACS Cliur (Becton Dickinson). CD40L stining. DO11.10 CD4 + T cells cultured with IL-18 (0 500 ng/ml) in the presence or sence of IL-2 (200 pm) for 4 dys were first treted with 10 µg/ml nti-fcγrii/iii for 30 min t 4 C nd then iotinylted nti-cd40l (Phrmingen) for 30 min t 4 C in stining uffer (PBS, 1% FCS). Cells were then wshed twice nd stined with FITC-nti-CD4 nd PE-leled vidin (Phrmingen) for 30 min. Smples were nlyzed on FACS Cliur (Becton Dickinson, Sn Jose). Ptients. 33 ptients hd LL, six hd TL nd six elonged to the IG. All ptients gve informed consent to this study, which ws pproved y our institutionl ord. We mesured serum IL-12 nd IL-18 in LL ptients efore (n = 33) or fter (n = 16) tretment with ntileprosy drugs nd in the ptients suffering from TL (n = 6) or elonging to the IG (n = 6). We lso mesured IL-12 nd IL-18 in ser from helthy sujects (n = 20). Assy for serum IL-12 nd IL-18. Serum smples were collected y venepuncture nd were stored t 80 C until nlysis. Serum IL-12 nd IL-18 concentrtions were mesured y commercilly ville IL-12 ELISA kit (Endogen, Wourn MA) nd IL-18 ELISA kit (MBL, Ngoy, Aichi, Jpn). Sttisticl nlysis. Results re geometric mens ± s.d. Student s t-test nd regression nlysis were used. P-vlues less thn 0.05 were ccepted s significnt. Acknowledgments We thnk Hyshir Biochemicl Lortories Inc. for providing recominnt murine IL-18, S.Akir nd K.Tked t Osk University for STAT6 / nd IL-18 -/ mice, M. Koyshi nd the Reserch Support Tem t Genetic Institute for murine sil-13ρα Fc nd H. Fukui for excellent technicl ssistnce. 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