REVIEW ARTICLERole of interleukin-18 in acute graft-vs-host disease

PAVAN REDDY and JAMES L. M. FERRARA

ANN ARBOR, MICHIGAN

Allogeneic hematopoietic cell transplantation (HCT) has emerged as an importanttherapeutic option for several malignant and nonmalignant diseases. In addition todelivering systemic chemoradiotherapy, the therapeutic potential of allogeneicHCT relies on the graft-vs-leukemia (GVL) effect, which eradicates residual malig-nant cells by way of immunologic mechanisms. Unfortunately, GVL effects areclosely associated with graft-vs-host disease (GVHD), the major complication ofallogeneic HCT. Separation of the toxicity of acute GVHD from the beneficial GVLeffects remains a major challenge to expanding the utility of this effective treatmentmodality. The pathophysiology of acute GVHD involves dysregulation of inflamma-tory cytokine cascades and donor T-cell responses to host alloantigens. Interleukin18 (IL-18) is a recently discovered cytokine with potent immunomodulatory effects.This unique cytokine has the capacity to induce Th1 or Th2 polarization, dependingon the immunologic context. The level of IL-18 is increased in acute GVHD, but thiscytokine’s role in the pathophysiology of acute GVHD is complex. It reduces theseverity of acute GVHD as a T helper 1 (Th1)-inducing cytokine when administeredearly after bone-marrow transplant to the lethally irradiated recipients. When ad-ministered to the donor, it can also reduce the severity of acute GVHD, as a T helper2 (Th2)-inducing cytokine. Despite reducing the severity of acute GVHD, IL-18preserves the GVL effect after bone-marrow transplant. Thus IL-18 has the remark-able capacity to modulate acute GVHD when administered either to the donor orthe recipient through distinct mechanisms. (J Lab Clin Med 2003;141:365-71)

Abbreviations: AICD � activation-induced cell death; BMT � bone-marrow transplantation;GVHD � graft-vs-host disease; GVL � graft-vs-leukemic; IFN � interferon; IL � interleukin; LPS �lipopolysaccharide; mAb � monoclonal antibody; MHC � major histocompatibility complex;NK � natural killer; STAT � signal transducer and activator of transcription; TNF � tumor necrosisfactor

A llogeneic BMT is most commonly used totreat various malignancies such as leuke-mias, lymphomas, multiple myeloma, and

renal-cell carcinoma. It represents the most potent formof immunotherapy against cancers by virtue of its GVLeffect.1 Unfortunately, the beneficial GVL effect istightly linked to acute GVHD, a potentially fatal com-plication of allogeneic BMT.2 The risk of GVHD mor-bidity and mortality has prevented wider and moreeffective application of this potent therapy. Under-standing the biology of GVHD and separating it frombeneficial GVL effects remains a major challenge.

Work in several laboratories, including ours, sug-gests that acute GVHD pathophysiology can be con-

From the Department of Internal Medicine, Division of Hematology/Oncology, Blood and Marrow Transplantation Program, Universityof Michigan Cancer Center.

Dr Reddy is a recipient of the Young Investigator Award from theAmerican Society of Clinical Oncology and the Outstanding YoungInvestigator from the Central Society for Clinical Research. Thiswork was supported by National Institutes of Health grant HL-03565to Dr Ferrara.

Submitted for publication November 7, 2002; accepted February 19,2003.

Reprint requests: Dr Pavan Reddy, University of Michigan CancerCenter, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0942; e-mail: reddypr@umich.edu.

Copyright © 2003 by Mosby, Inc. All rights reserved.

0022-2143/2003/$30.00 � 0

doi:10.1016/S0022-2143(03)00028-3

365

ceptualized in 3 sequential phases.2–4. In phase 1, theconditioning regimen (irradiation, chemotherapy, orboth) leads to damage and activation of host tissuesthrough the release of proinflammatory cytokines suchas TNF-� and IL-1.3–5 These cytokines can increase theexpression of MHC antigens and adhesion moleculeson host antigen-presenting cells, enhancing the recog-nition of host MHC and minor histocompatibility anti-gens by mature donor T-cells. Phase 2 is characterizedby the expansion of donor T-cells, secretion of cyto-kines that prime the mononuclear phagocytes, and in-duction of cytotoxic T-cell and NK-cell responses.Damage to the intestinal mucosa in phase 1 and bycytolytic effectors activated in phase 2 allows translo-cation of LPS from the intestinal lumen into the sys-temic circulation. Subsequently, LPS stimulates addi-tional cytokine production by the monocytes/macrophages and further promotes the inflammatoryresponse. In the effector phase (phase 3), the inflam-matory response, together with the cytotoxic T-lym-phocytes and NK cells, lead to target-tissue destructionin the BMT host. Damage to the gastrointestinal tract inphase 3 further increases LPS release, stimulating morecytokine production and tissue damage, thereby prop-agating the “cytokine storm” characteristic of acuteGVHD.2–4 In some experimental models, the “cytokinestorm” is amplified by donor Th1 cells and correlateswith the severity of acute GVHD, whereas a shift toTh2 polarization of donor cells reduces acute GVHD.4,5

The Th1/Th2 dichotomy of donor T-cells as it relates toacute GVHD, however, is not crisp.6

IL-18 was identified as a factor promoting IFN-�production.7 It is structurally related to IL-1, and itsreceptor resembles that of IL-1.8,9 IL-18 signals by wayof MyD88, which activates TNF receptor–associatedfactor and, ultimately, NF�B.10,11 Many cell types havebeen reported to produce IL-18, including macro-phages, T-cells, dendritic cells, Kupffer cells, astro-cytes, and microglia, intestinal and airway epithelialcells, keratinocytes, and osteoblasts; major targets ofIL-18 include macrophages, NK cells, T-cells, and B-cells.12,13

Serum levels of IL-18 are increased in human andexperimental models of acute GVHD after allogeneicBMT.15–19 A recent study demonstrated increased ex-pression of the IL-18 receptor on donor CD4� andCD8� cells in patients after allogeneic BMT that cor-related with the severity of acute GVHD. Okamoto etal14 demonstrated that IL-18 can reduce the severity ofchronic GVHD in experimental models, but its role inacute GVHD is not clear. Given the unique potential ofIL-18 to regulate both Th1 and Th2 responses12 and theimportance of these pathways in the development ofacute GVHD,20 our laboratory investigated the effect of

IL-18 administration on the development of acuteGVHD using well-established experimental mousemodels.2 All of the experiments performed conform to theUnit for Laboratory Animal Medicine’s ethical guidelinesfor the treatment of animals.

INHIBITION OF GVHD BY ADMINISTRATION OF IL-18TO BMT RECIPIENTS

We first made the observation that blockade of IL-18with mAb early in BMT after lethal radiation exacer-bated acute GVHD mortality.6 This was counterintui-tive, because IL-18 is a proinflammatory cytokine andserum levels of IL-18 are increased after BMT. Evenmore surprising, injection of 1 �g/mouse/day from day�2 to �2 significantly protected lethally irradiated F1mice that received bone marrow and splenic T-cellsfrom the MHC and multiple minor antigen–mis-matched B6 donor mice.6 Allogeneic recipients thatreceived anti–IL-18 mAb had significantly greaternumbers of total donor T-cells and CD4� cells in theirspleens during the first week compared with the controlgroup, suggesting that neutralization of IL-18 amplifiedthe donor T-cell response to host alloantigens, whereasIL-18 administration dampened the kinetics of donorT-cell expansion in the recipient spleens during the 5days after BMT.6 Because acute GVHD in this donor-recipient (parent into F1) strain combination is mainlydriven by CD4� donor T-cells,21 we also investigatedthe kinetics of CD4� and CD8� expansion and foundthat IL-18 administration primarily reduced the expan-sion of the CD4� T-cell subset.6

Role of Fas and IFN-�. The reduction in T-cell expan-sion in IL-18–treated mice was associated with agreater percentage of donor-cell apoptosis early afterBMT. In conjunction with the reduced donor T-cellexpansion and greater apoptosis, increased levels of Fasexpression were detected on donor CD4� cells.6 Thefunctional relevance of the increased Fas expressionand AICD by donor T-cells in IL-18–mediated protec-tion from GVHD was confirmed when IL-18 adminis-tration failed to protect the mice that received BMTfrom Fas-deficient donors. Thus, IL-18 protected miceagainst acute GVHD in a Fas-dependent manner. IFN-�is important in regulating the death of activated CD4�T lymphocytes22-26 and mediates the biologic effects ofIL-18.12 Serum levels of IFN-� were higher in theallogeneic recipients treated with IL-18, suggesting en-hanced Th1 polarization.6 Therefore, to determine therole of IFN-� in IL-18–mediated protection againstacute GVHD, we used IFN-�–deficient (GKO) mice asdonors. All mice receiving GKO donor cells died,whether or not they were treated with IL-18. Analysisof T-cell expansion (day �3) demonstrated equivalentexpansion of GKO donor T-cells in recipient spleens,

J Lab Clin Med366 Reddy and Ferrara June 2003

irrespective of IL-18 treatment, demonstrating a criticalrole for IFN-�.6 Therefore the ability of IL-18 to causedonor T-cell apoptosis and reduce the severity of acuteGVHD depended, at least in part, on donor-derivedIFN-�.

IL-18 and GVL. Because IL-18 enhances the cytolyticactivity of T-cells and has been shown to provideantitumor immunity,12,27,28 we also sought to determinewhether IL-18 could preserve the GVL effect conferredby donor T-cells after allogeneic BMT. Analysis ofsplenic T-cell function on day 14 after BMT demon-strated that IL-18 treatment did not affect the prolifer-ative responses or the cytolytic effector functions ofT-cells.29 When animals were injected with host-typeP815 mastocytoma cells at the time of BMT, all syn-geneic BMT recipients died as a result of systemicexpansion and infiltration of P815. The allogeneicBMT recipients effectively rejected their P815 cells,and IL-18 significantly reduced GVHD-related mortal-ity.29 The GVL effectors in this tumor model are theCD8� cells. Further examination of the CD8� cellcytotoxic pathways involved in this GVL effect dem-onstrated that perforin is critical.21,29 Taken together,these data demonstrate that IL-18 can attenuate acuteGVHD without impairing in vitro cytolytic function orperforin-dependent GVL activity in vivo after alloge-neic BMT.

These results are surprising because an increase inserum levels of IL-18 in acute GVHD initially sug-gested that this protein amplifies the cytokine dysregu-lation that characterizes this disorder. Our data demon-strate that IL-18 plays a far more complex role.Increased apoptosis of donor T-cells in the recipients ofallogeneic (vs syngeneic) BMT suggests that an attemptat such regulation may indeed occur normally during aGVHD reaction and that there is a natural contractionof the donor T-cell response to host alloantigens thatfollows immediately upon its rapid activation. Thiscontraction requires Fas expression and Th1 cytokinesecretion leading to peripheral deletion of alloreactivedonor T-cells by “fratricide,” which is enhanced byIL-18. Thus administration of IL-18 to the recipientmice early in BMT enhances Th1 polarization (IFN-�)and reduces GVHD by promoting Fas-dependent AICDof donor T-cells.

Similar inhibition of GVHD has been noted with theadministration of other Th1 cytokines, such as IFN-�,IL-2, and IL-12.30–34 IL-18 administration appears toimpart some of its beneficial effects in a manner similarto that of IL-12 (eg, attenuation of early donor T-cellexpansion and requirement of Fas expression andIFN-� production by donor cells.33,35,36 However, theydiffer in several respects: Blockade of IL-18 increasedGVHD-related mortality, unlike IL-12 blockade29,37;

the biphasic effect on serum IFN-� levels reported withIL-12 treatment was not observed with IL-18 treatment;29,38 and IL-18 treatment caused a decrease in serumTNF-�, but IL-12 treatment did not.29,39 Furthermore,it has been demonstrated that IL-12 from both donorand recipient sources contributes to increased severityof acute GVHD.40 Although a recent study suggeststhat the increased levels of serum IL-18 observed afterallogeneic BMT is likely host-derived, it did not ad-dress the role of IL-18 in GVHD pathophysiology.19

Therefore it is still not known whether host- or donor-derived IL-18 is critical for GVHD. Furthermore, it isalso possible that, as with IL-12, prolonged or delayedadministration of IL-18 has alternate effects on GVHDseverity.37,41

In a recent study, Arnold et al used IL-18–bindingprotein, a soluble IL-18 decoy receptor, to neutralizeIL-18 in a nonirradiated model of acute GVHD andobserved no change in GVHD severity.42 They con-cluded that IL-18 expression correlates with GVHD butthat its effect is redundant for the initiation and prop-agation of acute GVHD. In that study, the authorsobserved an increase in donor T-cell expansion afterIL-18 neutralization, but, in contrast to our findings, itdid not aggravate the disease.42 This may have been theresult of differences in conditioning between studies(although the type of antibodies used may also havebeen a factor). Furthermore, both of these studies areconsistent with a changing role of donor-derived IFN-�on acute GVHD with respect to intensity of condition-ing: The absence of donor-derived IFN-� is protectiveafter sublethal total-body irradiation, whereas its ab-sence after lethal irradiation is deleterious in CD4�-mediated GVHD.43

IMPACT ON GVHD OF IL-18 ADMINISTRATION INDONORS

IL-18 induces the secretion of several hematopoieticgrowth factors, such as granulocyte-colony–stimulatingfactor, granulocyte-macrophage colony–stimulatingfactor, and IL-6.12,44 Because IL-18 is nontoxic and caninduce hematopoietic growth factor secretion and maytherefore be suitable for administration to normal do-nors, we investigated whether the modulation of donorT-cell responses with IL-18 before BMT would regu-late acute GVHD and GVL responses.44–47

Recipient B6 mice were lethally irradiated and trans-planted with T-cell–depleted bone marrow and splenicT-cells from donor BALB/c mice pretreated with 1�g/day of recombinant murine IL-18 or diluent for 10days. Animals that received allogeneic T-cells fromIL-18–pretreated donors showed significantly bettersurvival and less clinical GVHD after BMT than thecontrol mice.

J Lab Clin MedVolume 141, Number 6 Reddy and Ferrara 367

In contrast to the administration of IL-18 to recipientmice early in BMT, which reduced acute GVHD byattenuating donor T-cell expansion, IL-18 treatment ofdonors did not significantly alter donor T-cell expan-sion in the recipient spleens in the 2 weeks afterBMT.29 Because IL-18 can promote either Th1 or Th2polarization, depending on the context,12,45,48,49 weevaluated the effects of IL-18 administration on cyto-kine secretion of donor T-cells after BMT. IL-18–treated donor T-cells secreted significantly less IFN-�and more IL-4 in mixed lymphocyte reaction culturesafter BMT.55 Furthermore, animals that received allo-geneic T-cells from donors pretreated with IL-18 hadsignificantly reduced levels of serum IFN-� afterBMT.55 Because GVHD in this model is predominantlymediated by CD4� cells, additional experiments withCD4� with T-cell–depleted bone marrow from IL-18–treated donors also reduced the severity of acute GVHDmortality.35,55 Thus IL-18 administration in the donorsproduced a diminished Th1 response and an enhancedTh2 response after BMT, in stark contrast to enhancedTh1 response on administration to the recipient earlyafter BMT.

Role of IFN-�, STAT 4, and STAT 6. Because the pre-treatment of donors with IL-18 resulted in a decrease inIFN-� and because Th1 responses of donor T-cells areknown to be critical to GVHD induction,2 we nextdetermined whether donor-derived IFN-� might be crit-ical for IL-18 to mediate its effect. Surprisingly, whenIFN-�–deficient mice were pretreated with rmIL-18and used as donors, the protective effect of IL-18 wasagain preserved, thereby demonstrating that the ab-sence of IFN-� secretion by donor T-cells is not criticalto IL-18–mediated GVHD protection.55 To confirmthese surprising results and to elucidate the molecularmechanism of the effect of IL-18 on donor T-cellpolarization and GVHD protection, we used donormice that lacked the STAT4 and that had impaired Th1responses.50,51 IL-18 treatment of BALB/c STAT4-deficient donor mice reduced GVHD in B6 recipients,confirming that STAT4 is not required for the protec-tive effect of IL-18. Together, these two separate ex-periments confirm each other’s findings and show thatthe alteration in Th1 response as determined by secre-tion of IFN-� is not critical to the GVHD protectionmediated by IL-18.

Pretreatment of wild-type donors with IL-18 alsoenhanced secretion of IL-4 by donor T-cells after BMT.Yoshimoto et al recently demonstrated that STAT6 iscritical for IL-18–mediated Th2 polarization.45 Wetherefore determined whether STAT6 signaling, whichis critical to Th2 responses, is important for the protec-tive effect of IL-18 on GVHD.50,51 In contrast to itseffects on wild-type donors, injection of IL-18 in-

creased IFN-� but not IL-4 in the sera of BALB/cSTAT6-deficient mice and was not able to confer pro-tection against acute GVHD mortality on B6 recipi-ents.52 These data therefore support the hypothesis thatalteration of Th2 responses by donor T-cells by way ofSTAT6 signaling is critical to the protective effect ofIL-18. Taken together, these data suggest that, in starkcontrast to the mechanism of IL-18’s effect when in-jected into the recipients, the alteration in Th1 responseas determined by the secretion of IFN-� is not criticalfor IL-18–mediated GVHD protection.

Effect on GVL. We also determined whether pretreat-ment of donor mice preserved CD8�-mediated GVLby utilizing the EL-4, an MHC II-/- lymphoma. In thismodel, GVH is primarily mediated by donor CD4�and GVL is mediated by CD8� cells.36 All syngeneicrecipients exhibited 100% mortality from EL-4 tumorinfiltration, regardless of IL-18 treatment of the donormice. However, recipients of transplants from IL-18–treated allogeneic BMT donors showed significantlybetter survival than the controls, suggesting that allo-geneic BMT from IL-18–treated donors reduces acuteGVHD severity while preserving a GVL effect.52

DISCUSSION

IL-18 is the only cytokine that has the remarkablecapacity to induce either Th1 or Th2 polarization, de-pending on the stimulatory context and the immuno-logic milieu.12 We have demonstrated that administra-tion of IL-18 to BMT hosts early after transplantattenuates acute GVHD by enhancing Fas-dependentapoptosis of donor T-cells and that donor-derivedIFN-� is critical for this protection.6 In this context, thepredominant effect of IL-18 is to induce Th1 cytokines,which are critical for its GVHD protection. By contrast,administration of IL-18 to the donors protects againstacute GVHD mortality byway of a seemingly oppositeeffect: use of donors that lack IFN-� and STAT4 re-tained the protection of IL-18 treatment, showing thatalterations in Th1 cytokines play a less important rolein this situation. IL-18 lost its GVHD-protective effect,however, when STAT6 deficient mice were used asdonors, suggesting that enhanced Th2 cytokine produc-tion by way of STAT6 is critical to this effect.52 Ourdata therefore demonstrate that IL-18 plays a variedrole in modulating cytokine responses and Th polariza-tion, depending on whether the donor T-cells are rest-ing or have already been activated by alloantigens. It isalso noteworthy that a single cytokine such as IL-18can bring about similar outcomes in a complex in vivoimmunologic reaction such as acute GVHD by way ofdistinct mechanisms depending on whether it is givento the donor or to the recipient.

The cellular and molecular basis for tolerance relies

J Lab Clin Med368 Reddy and Ferrara June 2003

on several different mechanisms that are not necessarilymutually exclusive and that are probably complemen-tary. These mechanisms include both deletional mech-anisms in which donor-reactive clones are destroyedand nondeletional regulation of immune cells, includ-ing clonal anergy, immune deviation toward Th2, andactive suppression of donor-reactive cells.53,54 Thecomplex and at times paradoxical role of IL-18 admin-istered to BMT recipients may thus be explained by theintricate dynamics of donor T-cell activation, expan-sion and contraction by AICD (ie, deletional tolerance).IL-18 administration early after BMT mediates GVHDreduction by peripheral deletion of alloreactive donorT-cells by way of the Fas/FasL pathway in a Th1-dependent manner. Although moderate amounts ofIL-18 may amplify GVHD through induction of IFN-�in the recipients of intense conditioning, extremes (ei-ther high or low) of IL-18 (other Th1 cytokines) mayhasten death of activated donor CD4� T cells. Partic-ularly during T-cell expansion shortly after BMT,IL-18 aborts the expansion of alloreactive T-cells andthus reduces GVHD. In contrast to this deletionalmechanism, pretreatment of donors with IL-18 protectsagainst GVHD by way of immune deviation, a nonde-letional mechanism.

These data demonstrate the critical and unique role ofIL-18 in modulating acute GVHD by contrasting toler-ance mechanisms and underscore the importance andcomplexity of the role Th1/Th2 polarization in GVHD.Given the pleiotropic and the compensatory nature ofIL-18’s effect on immune modulation, it is probablethat IL-18 has effects that vary not only with the timingand dosage of administration but also with the condi-tioning used for BMT and with the donor/recipientstrain combination. Our studies have been concentratedon the role of IL-18 in GVHD models that differ acrossboth major and minor histocompatibility antigens, andexamination of the effects of IL-18 on GVHD elicitedby isolated MHC class II or I antigens should alsoprove informative. Furthermore, because T-cell polar-ization independent of STAT (4 and 6) molecules isalso possible, it remains to be determined whetherSTAT independent Th1/2 induction also plays a role inthe effects of IL-18.50,55

IL-18, as a Th1-promoting cytokine, has been shownto be critical to protection from various viral (herpessimplex virus, vaccinia, murine cytomegalovirus, andhuman papillomavirus), bacterial (tuberculosis, Shi-gella, and Salmonella), protozoan (malaria and leish-mania) and fungal (cryptococcal, Aspergillus) infec-tions.12 However IL-18 also appears to promote thedevelopment of characteristic Th2 response of lepro-matous leprosy after infection with Mycobacterium lep-rae.45 Similarly, the effects of IL-18 on airway inflam-

mation are variable because the cytokine can promoteeither a Th1 or Th2 response; for example, it acts as aTh1 promoter in airway-hyperreactivity models andsarcoidosis but has also been shown to increase eosin-ophil accumulation in the airways (Th2 promoter) in acockroach-allergen model.56,57 All of these data furtherunderscore the complex and unique role of IL-18 in theinduction of Th1/Th2 polarization in a variety of im-munologic processes besides acute GVHD.

Therefore, better understanding of the interactions ofIL-18 in the cytokine cascades of after allogeneic BMTwill shed light on the complex role of T-cell polariza-tion in GVHD pathophysiology and may lead to noveltherapeutic strategies for this toxic complication ofallogeneic BMT.

REFERENCES

1. Appelbaum FR. Haematopoietic cell transplantation as immuno-therapy. Nature 2001;411:385-9.

2. Hill GR, Ferrara JL. The primacy of the gastrointestinal tract asa target organ of acute graft-versus-host disease: rationale for theuse of cytokine shields in allogeneic bone marrow transplanta-tion. Blood 2000;95:2754-9.

3. Ferrara JLM, Deeg HJ. Graft versus host disease. N Engl J Med1991;324:667-74.

4. Goker H, Haznedaroglu IC, Chao NJ. Acute graft-vs-host dis-ease: pathobiology and management. Exp Hematol 2001;29:259-77.

5. Antin JH, Ferrara JLM. Cytokine dysregulation and acute graft-versus-host disease. Blood 1992;80:2964-8.

6. Reddy P, Teshima T, Kukuruga M, Ordemann R, Liu C, LowlerK, et al. Interleukin-18 regulates acute graft-versus-host diseaseby enhancing Fas-mediated donor T cell apoptosis. J Exp Med2001;194:1433-40.

7. Okamura H, Tsutsui H, Komatsu T, Yutsudo M, Hakura A,Tanimoto T, et al. Cloning of a new cytokine that inducesIFN-gamma production by T cells. Nature 1995;378:88-91.

8. Bazan J, Timans J, Kastelein R. A newly defined interleukin-1?Nature 1996;379:591.

9. Hoshino K, Tsutsui H, Kawai T, Takeda K, Nakanishi K, TakedaY, et al. Cutting edge: generation of IL-18 receptor-deficientmice: evidence for IL-1 receptor-related protein as an essentialIL-18 binding receptor. J Immunol 1999;162:5041-4.

10. Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sak-agami M, et al. Targeted disruption of the MyD88 gene results inloss of IL-1 and IL-18–mediated function. Immunity 1998;9:143-50.

11. Akira S. Role of IL-18 in innate immunity. Curr Opin Immunol2000;12:59-63.

12. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleu-kin-18 regulates both th1 and th2 responses. Annu Rev Immunol2001;19:423-74.

13. Swain SL. Interleukin 18: tipping the balance towards a T helpercell 1 response. J Exp Med 2001;194:F11-4.

14. Okamoto I, Kohno K, Tanimoto T, Iwaki K, Ishihara T, Aka-matsu S, et al. IL-18 prevents the development of chronic graft-versus-host disease in mice. J Immunol 2000;164:6067-74.

15. Fujimori Y. Elevated interleukin (IL)-18 levels during acutegraft-versus-host disease after allogeneic bone marrow transplan-tation. Br J Haematol 2000;109:652-7.

16. Nakamura H, Komatsu K, Ayaki M, Kawamoto S, Murakami M,

J Lab Clin MedVolume 141, Number 6 Reddy and Ferrara 369

Uoshima N, et al. Serum levels of soluble IL-2 receptor, IL-12,IL-18, and IFN-gamma in patients with acute graft-versus-hostdisease after allogeneic bone marrow transplantation. J AllergyClin Immunol 2000;106:S45-50.

17. Zecchina G, Novick D, Rubinstein M, Barak V, Dinarello C,Nagler A. Interleukin-18 binding protein in acute graft versushost disease and engraftment following allogeneic peripheralblood stem cell transplants. J Hematother Stem Cell Res 2001;10:769-76.

18. Hu HZ, Li GL, Lim YK, Chan SH, Yap EH. Kinetics of inter-feron-gamma secretion and its regulatory factors in the earlyphase of acute graft-versus-host disease. Immunology 1999;98:379-85.

19. Itoi H, Fujimori Y, Tsutsui H, Matsui K, Futatsugi S, OkamuraH, et al. Fas ligand–induced caspase-1–dependent accumulationof interleukin-18 in mice with acute graft-versus-host disease.Blood 2001;98:235-7.

20. Hill G, Ferrara J. The primacy of the gastrointestinal tract as atarget organ of acute graft-versus-host disease: rationale for theuse of cytokine shields in allogeneic bone marrow transplanta-tion. Blood 2000;95:2754-9.

21. Teshima T, Hill G, Pan L, Brinson Y, van den Brink MR, CookeK, et al. IL-11 separates graft-versus-leukemia effects from graft-versus-host disease after bone marrow transplantation. J ClinInvest 1999;104:317-25.

22. Liu Y, Janeway CA Jr. Interferon gamma plays a critical role ininduced cell death of effector T cell: a possible third mechanismof self-tolerance. J Exp Med 1990;172:1735-9.

23. Novelli F, Bernabei P, Ozmen L, Rigamonti L, Allione A, PestkaS, et al. Switching on of the proliferation or apoptosis of acti-vated human T lymphocytes by IFN-gamma is correlated withthe differential expression of the alpha- and beta-chains of itsreceptor. J Immunol 1996;157:1935-43.

24. Dalton DK, Haynes L, Chu CQ, Swain SL, Wittmer S. Interferongamma eliminates responding CD4 T cells during mycobacterialinfection by inducing apoptosis of activated CD4 T cells. J ExpMed 2000;192:117-22.

25. Siegel RM, Chan FK, Chun HJ, Lenardo MJ. The multifacetedrole of Fas signaling in immune cell homeostasis and autoim-munity. Nat Immunol 2000;1:469-74.

26. Refaeli Y, Van Parijs L, Alexander SI, Abbas AK. Interferongamma is required for activation-induced death of T lympho-cytes. J Exp Med 2002;196:999-1005.

27. Osaki T, Peron J-M, Cai Q, Okamura H, Robbins P, KurimotoM, et al. IFN gamma inducing factor/IL-18 administration me-diates IFN gamma and IL-12 independent antitumor effects.J Immunol 1998;160:1742-9.

28. Heuer JG, Tucker-McClung C, Hock RA. Neuroblastoma cellsexpressing mature IL-18, but not proIL-18, induce a strong andimmediate antitumor immune response. J Immunother 1999;22:324-35.

29. Reddy P, Teshima T, Hildebrandt G, Duffner U, Maeda Y,Cooke KR, et al. Interleukin 18 preserves a perforin-dependentgraft-versus-leukemia effect after allogeneic bone marrow trans-plantation. Blood 2002;100:3429-31.

30. Brok HPM, Heidt PJ, van der Meide PH, Zurcher C, Vossen JM.Interferon-� prevents graft-versus-host disease after allogeneicbone marrow transplantation in mice. J Immunol 1993;151:6451-9.

31. Brok HPM, Vossen JM, Heidt PJ. Interferon-�–mediated pre-vention of graft-versus-host disease: development of immunecompetent and allo-tolerant T cells in chimeric mice. BoneMarrow Transplant 1997;19:601-6.

32. Sykes M, Romick ML, Sachs DH. Interleukin 2 prevents graft-

versus-host disease while preserving the graft-versus-leukemiaeffect of allogeneic T cells. Proc Natl Acad Sci U S A 1990;87:5633-7.

33. Dey BR, Yang YG, Szot GL, Pearson DA, Sykes M. Interleu-kin-12 inhibits graft-versus-host disease through an Fas-medi-ated mechanism associated with alterations in donor T-cell acti-vation and expansion. Blood 1998;91:3315-22.

34. Sykes M, Pearson DA, Taylor PA, Szot GL, Goldman SJ, BlazarBR. Dose and timing of interleukin (IL)-12 and timing and typeof total-body irradiation: effects on graft-vs.-host disease inhibi-tion and toxicity of exogenous IL-12 in murine bone marrowtransplant recipients. Biol Blood Marrow Transplant 1999;5:277-84.

35. Yang YG, Dey BR, Sergio JJ, Pearson DA, Sykes M. Donor-derived interferon gamma is required for inhibition of acutegraft-versus-host disease by interleukin 12. J Clin Invest 1998;102:2126-35.

36. Yang YG, Sykes M. The role of interleukin-12 in preserving thegraft-versus-leukemia effect of allogeneic CD8 T cells indepen-dently of GVHD. Leuk Lymphoma 1999;33:409-20.

37. Williamson E, Garside P, Bradley JA, More IAR, Mowat AM.Neutralizing IL-12 during induction of murine acute graft-ver-sus-host disease polarizes the cytokine profile toward a Th2-typealloimmune response and confers long term protection fromdisease. J Immunol 1997;159:1208-15.

38. Sykes M, Szot GL, Nguyen PL, Pearson DA. Interleukin-12inhibits murine graft-versus-host disease. Blood 1995;86:2429-38.

39. Yang YG, Sergio JJ, Pearson DA, Szot GL, Sykes M. Interleukin12 preserves the graft-vs-leukemia effect of allogeneic T cellswhile inhibiting graft-vs-host disease. Blood 1997;90:4651-60.

40. Welniak LA, Blazar BR, Wiltrout RH, Anver MR, Murphy WJ.Role of interleukin-12 in acute graft-versus-host disease. Trans-plant Proc 2001;33:1752-3.

41. Williamson E, Garside P, Bradley JA, Mowat AM. IL-12 is acentral mediator of acute graft-versus-host disease in mice. J Im-munol 1996;157:689-99.

42. Arnold D, Wasem C, Juillard P, Graber P, Cima I, Frutschi C, etal. IL-18–independent cytotoxic T lymphocyte activation andIFN-gamma production during experimental acute graft-versus-host disease. Int Immunol 2002;14:503-11.

43. Welniak LA, Blazar BR, Anver MR, Wiltrout RH, Murphy WJ.Opposing roles of interferon-gamma on CD4� T cell-mediatedgraft-versus-host disease: effects of conditioning. Biol BloodMarrow Transplant 2000;6:604-12.

44. Ogura T, Ueda H, Hosohara K, Tsuji R, Nagata Y, KashiwamuraS, et al. Interleukin-18 stimulates hematopoietic cytokine andgrowth factor formation and augments circulating granulocytesin mice. Blood 2001;98:2101-7.

45. Yoshimoto T, Mizutani H, Tsutsui H, Trauth N, Yamanaka K,Tanaka M, et al. IL-18 induction of IgE: dependence on CD4�T cells, IL-4 and STAT-6. Nat Immunol 2000;1:132-7.

46. Carson W, Dierksheide J, Jabbour S, Anghelina M, BouchardP, Ku G, et al. Coadministration of interleukin-18 and inter-leukin-12 induces a fatal inflammatory response in mice:critical role of natural killer cell interferon gamma productionand STAT-mediated signal transduction. Blood 2000;96:1465-73.

47. Okamoto M, Kato S, Oizumi K, Kinoshita M, Inoue Y, HoshinoK, et al. Interleukin 18 (IL-18) in synergy with IL-2 induceslethal lung injury in mice: a potential role for cytokines, chemo-kines, and natural killer cells in the pathogenesis of interstitialpneumonia. Blood 2002;99:1289-98.

48. Hoshino T, Yagita H, Ortaldo JR, Wiltrout RH, Young HA. In

J Lab Clin Med370 Reddy and Ferrara June 2003

vivo administration of IL-18 can induce IgE production throughTh2 cytokine induction and up-regulation of CD40 ligand(CD154) expression on CD4� T cells. Eur J Immunol 2000;30:1998-2006.

49. Hoshino T, Kawase Y, Okamoto M, Yokota K, Yoshino K,Yamamura K, et al. Cutting edge: IL-18–transgenic mice: invivo evidence of a broad role for IL-18 in modulating immunefunction. J Immunol 2001;166:7014-8.

50. Glimcher LH, Murphy KM. Lineage commitment in the immunesystem: the T helper lymphocyte grows up. Genes Dev 2000;14:1693-711.

51. Robinson DS, O’Garra A. Further checkpoints in Th1 develop-ment. Immunity 2002;16:755-8.

52. Reddy P, Teshima T, Hildebrandt G, Williams DL, Liu C, CookeKR, et al. Pretreatment of donors with interleukin-18 attenuates

acute graft-versus-host disease via STAT6 and preserves graft-versus-leukemia effects. Blood 2003;101:2877-85.

53. Kamradt T, Mitchison NA. Tolerance and autoimmunity. N EnglJ Med 2001;344:655-64.

54. Li XC, Strom TB, Turka LA, Wells AD. T cell death andtransplantation tolerance. Immunity 2001;14:407-16.

55. Farrar JD, Asnagli H, Murphy KM. T helper subset development:roles of instruction, selection, and transcription. J Clin Invest2002;109:431-5.

56. Gracie AJ, Robertson SE, McInnes IB. Interleukin-18. J LeukocBiol 2003;73:213-24.

57. Campbell E, Kunkel SL, Strieter RM, Lukacs NW. Differentialroles of IL-18 in allergic airway disease: induction of eotaxin byresident cell populations exacerbates eosinophil accumulation.J Immunol 2000;164:1096-102.

J Lab Clin MedVolume 141, Number 6 Reddy and Ferrara 371