UNIT 6.26Measurement of Human and MouseInterleukin 18

IL-18, originally designated as interferon γ– (IFN-γ)-inducing factor (IGIF), is a pleiot-ropic cytokine secreted by activated macrophages and Kupffer cells (Okamura et al., 1995,1998; Dinarello, 1999; Nakanishi et al., 2001a,b). The major activity associated with thiscytokine is induction of IFN-γ production from T cells, B cells, and NK cells, especiallyin collaboration with IL-12 (Okamura et al., 1995; Yoshimoto et al., 1998; Tsutsui et al.,1997). However, without IL-12, IL-18 shows the opposite action and induces IL-4 andIL-13 production from T cells and NK cells (Yoshimoto et al., 1999, 2000; Hoshino etal., 1999, 2000).

IL-18 is synthesized as a 24-kDa precursor molecule without a signal peptide, and needsto be enzymatically cleaved to become active. Caspase-1 cleaves pro-IL-18 at asparticacid in the P1 position, yielding the mature 18-kDa peptide IL-18 (Gu et al., 1997).However, pro-IL-18 is also processed by caspases other than caspase-1 (Tsutsui et al.,1999). Murine macrophages (e.g., Kupffer cells) secrete functional IL-18 only afteractivation with appropriate stimuli, including lipopolysaccharide (LPS), CD40 ligand (L),or FasL (Okamura et al., 1995; Tsutsui et al., 1997, 1999). In contrast, human peripheralblood mononuclear cells (PBMC) can secrete pro-IL-18 under normal condition (Purenet al., 1999). Therefore, it is important to determine whether the produced IL-18 is anactive or precursor form.

The usefulness of measuring IL-18 in the sera of patients has been reported by the authorsof this unit and other investigators. For example, the authors (Fujimori et al., 2000) andothers (Nakamura et al., 2000) have shown that patients with acute graft-versus-hostdisease (GVHD) have high levels of IL-18 in their sera, which subsided after appropriatetreatment (Fujimori et. al., 2000). These results suggest that IL-18 may play importantroles in the pathogenesis of acute GVHD and that measurement of serum IL-18 levelscan be useful indicator of acute GVHD. In addition, we have shown that patients withlepromatous leprosy, but not those with tuberculoid leprosy, show a high serum level ofIL-18, suggesting involvement of IL-18 in the development of Th2-immune responses(Yoshimoto et al., 2000).

This unit describes functional assays for measurement of bioactive human and mouseIL-18 (see Basic Protocol 1) and ELISAs for measurement of murine and human IL-18proteins (see Basic Protocol 2). The functional assays (see Basic Protocol 1) are basedon the induction of IFN-γ production by IL-18 (Yoshimoto et al., 1998; Tominaga et al.,2000). Basic Protocol 2 is an ELISA that measures the concentration of human or mouseIL-18. Using a combination of monoclonal antibodies against human or mouse IL-18, theproform and/or mature form of IL-18 can be detected by ELISA. In this protocol, ELISAfor human IL-18 is specific for active IL-18; however ELISA for murine IL-18 detectsboth forms. The authors recommend that investigators measure solutions containing IL-18by a combination of functional bioassay (see Basic Protocol 1) and ELISA (see BasicProtocol 2) to prove that measured protein is a bioactive one.

NOTE: All protocols using live animals must first be reviewed and approved by anInstitutional Animal Care and Use Committee (IACUC) or must conform to governmentalregulations regarding the care and use of laboratory animals.

Supplement 44

Contributed by Tomohiro Yoshimoto, Hiroko Tsutsui, Haruki Okamura, and Kenji NakanishiCurrent Protocols in Immunology (2001) 6.26.1-6.26.12Copyright © 2001 by John Wiley & Sons, Inc.

6.26.1

Cytokines andTheir CellularReceptors

BASICPROTOCOL 1

IFN-γ INDUCTION ASSAY TO QUANTITATE MOUSE AND HUMAN IL-18IL-18 and IL-12 promptly and synergistically induce T cells to develop into IFN-γ-pro-ducing cells (Yoshimoto et al., 1998). TCR engagement or cyclosporin A treatment doesnot affect IFN-γ production. Pretreatment of T cells with IL-12 renders them responsiveto IL-18, which induces IFN-γ production, while IL-18-pretreated T cells do not produceIFN-γ as a result of subsequent stimulation with IL-12. Therefore, T cell IFN-γ productionis effectively induced by simultaneous or sequential stimulation with IL-12 and IL-18.These IL-12-stimulated T cells increase the amount of IL-18R α chain (Yoshimoto et al.,1998; Tominaga et al., 2000). In this protocol, measurement of the action of mouse orhuman IL-18 by costimulation of T cells with IL-12 is described. While this assay issemiquantitative, it is superior to the more quantitative techniques described in this unitfor cases where it is important to determine whether the test fluid contains biologicallyactive IL-18. The protocol described here uses mouse T cells for measuring mouse IL-18.Similar steps can be used with human T cells for measuring human IL-18, with only minordifferences as noted in annotations to the steps involved.

Materials

Mice (BALB/c or C57BL/6 strain)Anti-asialo GM1 antibody (Wako)Mouse IL-12 (e.g., R & D Systems)Mouse IL-18 reference standard (commercially available from MBL International)Complete RPMI-10 medium (see recipe)Unknown sample for assay of IL-18Mouse or human IFN-γ ELISA kits (e.g., R & D Systems)

17 × 100-mm polystyrene tubes96-well flat-bottom microtiter plates with lids (e.g. Costar)24-well plates (e.g., Falcon)Multichannel and repeating pipettors

Additional reagents and supplies for intravenous injection of mice (UNIT 1.6) andpreparing mouse splenic T cells (UNIT 3.2)

NOTE: All solutions and equipment coming into contact with cells must be sterile, andaseptic technique should be used accordingly.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubatorunless otherwise indicated.

Prepare mouse T cell suspension containing IL-121. Inject mice intravenously with 400 µg anti–asialo GM1 antibody 3 days before they

are to be used as a source of spleen cells for T cell purification.

For human T cell preparation, PBMC are prepared as in the Basic Protocol of UNIT 7.1 ormononuclear cells are prepared from tonsillar tissue as in UNIT 7.8. Monocytes/macro-phages are removed by adherence to plastic (Support Protocol 1 in UNIT 7.1), and B cellsare depleted by adherence to nylon wool (Basic Protocol 1 in UNIT 7.7). NK cells are depletedby treatment with leucine methyl ester as described in Support Protocol 2 of UNIT 7.1, exceptthat leucine methyl ester is used at a final concentration of 5 mM. Human T cells may alsobe purified by negative immunomagnetic selection as described in UNIT 7.4.

2. Prepare a 2 × 106 cells/ml suspension of mouse splenic T cells (UNIT 3.2) in completeRPMI-10, in 17 × 100-mm polystyrene tubes. Add mouse IL-12 to a final concentra-tion of 20 ng/ml.

The dose of IL-12 is critical in this assay. For IL-18-dependent induction of mouse IFN-γproduction, costimulation of T cells with 20 ng/ml of mIL-12 is required. For IL-18-depend-

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Measurement ofHuman and

MouseInterleukin-18

ent induction of human IFN-γ production, costimulation of T cells with 100 ng/ml humanIL-12 (R & D Systems) is required.

For human T cells, the suspension is prepared at the same concentration as above.

Dilute IL-18 standard and samples3. Dilute mouse IL-18 reference standard to 100 ng/ml in 24-well plates using complete

RPMI-10 medium. Prepare four additional serial 5-fold dilutions (so that the lowestmouse IL-18 concentration is 0.16 ng/ml).

Human IL-18 reference standard is also commercially available from MBL International.For human cells, prepare four additional serial 2-fold dilutions of the initial 100 ng/ml, sothat the lowest concentration of human IL-18 is 6.25 ng/ml.

4. Prepare 2-fold serial dilutions of unknown sample in 24-well plates using completeRPMI-10.

5. Add 100 µl of each IL-18 reference standard and unknown sample dilution toduplicate or triplicate wells of 96-well flat-bottom plate. Add 100 µl of completeRPMI-10 alone to duplicate or triplicate wells of 96-well flat-bottom plate fordetermination of background.

6. Incubate plates 1 hr.

Culture T cells with IL-18 and IL-12 for IFN-γ production7. Using a repeating pipettor, add 100 µl of T cell suspension (2 × 105 cells/well)

containing IL-12 (step 1) to each well of the 96-well flat-bottom plate with IL-18standard and unknown samples (see steps 5 and 6).

IL-12-stimulated human T cells can respond to IL-1β. Thus, addition of 10 �g/ml anti-hu-man IL-1R type 1 monoclonal antibody (e.g., clone 4C1 from PharMingen) is needed inthe case of the human IL-18 bioassay. In contrast, IL-12-stimulated mouse T cells have nosuch capacity.

For mouse cells, activity detected in IL-18 assay can be shown to be due to IL-18 itself byaddition of 5 to 10 �g/ml neutralizing anti-IL-18 antibody such as that from clone 93-10C(MBL International) to appropriate control wells. For human cells, activity detected inIL-18 assay can be shown to be due to IL-18 itself by addition of 5 to 10 �g/ml neutralizinganti-IL-18 antibody such as that from clone 125-2H (MBL International) to appropriatecontrol wells.

8. Incubate the plates for 3 days (for mouse).

For human cells, plates are incubated for 4 days.

9. After incubation period is complete, centrifuge the plates 10 min at ∼250 × g in amicrotiter plate carrier, 4°C. Transfer 150 µl of each supernatant into a correspondingwell of a 96-well round bottom plate and store at −20°C until use.

Estimation of IL-18 activity by measuring mouse IFN-γ10. Measure IFN-γ in each supernatant by a commercially available ELISA kit for mouse

(or human) IFN-γ.

11. Construct a standard curve for IFN-γ production versus concentration of IL-18 usedfor stimulation. Calculate the concentration of IL-18 in the unknown samples usingthis standard curve.

To determine the concentration of IL-18 in unknown samples, the supernatant and a controlstandard, e.g., recombinant (r) IL-18, must be assayed at several dilutions. The dose of rIL-18that produces 50% of the IFN-γ production obtained with a saturating level of rIL-18 should

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Cytokines andTheir CellularReceptors

be determined first. Then, the IL-18 level in the test sample is estimated by multiplying thisvalue by the inverse of the dilution that produce 50% of the maximal level of IFN-γ.

For human T cell IFN-γ production, it is difficult to determine a saturating level of rIL-18to induce IFN-γ. Therefore, estimate IL-18 titer in the unknown sample dilution that resultsin a value of IFN-γ production that lies on the steep portion of the standard curve.

BASICPROTOCOL 2

ELISA FOR HUMAN AND MOUSE IL-18

The assay described in this protocol specifically measures IL-18. This ELISA is simplerto perform than the bioassay, which depends on the measurement of IFN-γ produced inIL-12-stimulated T cells (see Basic Protocol 1). However, some combinations of mono-clonal antibodies for human and murine IL-18 detect both pro- and mature-forms of IL-18.Therefore, it is important to use the specific monoclonal antibodies which can recognizeactive form of IL-18, or to test the samples in parallel with functional bioassay.

Human or mouse IL-18 is captured from IL-18-containing culture fluid or serum by amonoclonal antibody specific for human or mouse mature IL-18, after the monoclonalantibody has been absorbed to the wells of EIA plate. The test fluid is then washed fromthe wells, and captured IL-18 is detected using a peroxidase-conjugated monoclonalantibody specific for human or mouse IL-18.

Materials

ELISA coating antibody (see recipe) appropriate for human or mouse IL-18Coating buffer: PBS (APPENDIX 2A), pH 7.4Washing buffer: Dulbecco’s phosphate-buffered serum (D-PBS; e.g., Life

Technologies) containing 0.01% (v/v) Tween 20; store up to 1 week at roomtemperature

Blocking solution: Dulbecco’s phosphate-buffered serum (D-PBS; e.g., LifeTechnologies) containing 1% (w/v) bovine serum albumin (BSA); store up to 4weeks at 4°C

IL-18 reference standard (commercially available from MBL International)Unknown sample containing IL-18Assay buffer (see recipe)Conjugation buffer (see recipe)Peroxidase conjugated rat anti-human IL-18 monoclonal antibody (159-12B) or

anti-mouse IL-18 monoclonal antibody (93-10C): available from MBLInternational

Chromogenic substrate for detecting peroxidase: e.g., tetramethylbenzidine(TMB)/H2O2 (Becton Dickinson), prepared according to manufacturer’sinstructions

Stop solution appropriate for substrate employed (e.g., 2 N H2SO4)

Multichannel and repeating pipettors96-well EIA plates with plate sealers (e.g., EIA/RIA plate from Costar)ELISA plate reader (e.g., Benchmark, Bio-Rad) with appropriate filter for

peroxidase substrate employed

Coat plate with monoclonal anti-IL-18 antibody and block1. Thaw aliquot of appropriate ELISA coating antibody stock. If anti-human IL-18

antibody (clone 125-2H) is being used, dilute to 5 µg/ml in coating buffer; ifanti-mouse IL-18 antibody (clone 74) is being used, dilute to 15 µg/ml in coatingbuffer.

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MouseInterleukin-18

2. Using a multichannel pipettor, immediately add 100 µl diluted coating antibody toeach well of 96-well EIA plate. Seal plate with plate sealer and incubate overnightat 4°C.

3. Wash plate four times by repeatedly aspirating or flicking out well contents and fillingwells with washing buffer.

4. Add 200 µl blocking solution to each well and incubate either 1 hr at 37°C orovernight at 4°C.

Antibody-coated plates for human and mouse IL-18 are commercial available from MBLInternational.

Capture IL-18 on antibody-coated plate5. Dilute IL-18 reference standard to 1000 pg/ml in assay buffer. Prepare five additional

serial 2-fold dilutions in 1.5-ml microcentrifuge tubes (so that the lowest IL-18concentration is 31.2 pg/ml).

6. Dilute each sample with assay buffer. Using 1.5-ml microcentrifuge tubes, prepare5-fold serial dilutions of unknown samples in assay buffer, using a starting dilutionthat will allow a range of IL-18 concentrations from 1000 pg/ml to 100 pg/ml.

For example, human or mouse serum is diluted 1:4 with assay buffer by adding 50 �l ofsample to 200 �l of assay buffer. Sample dilution may vary between different specimens.Appropriate sample dilution should be established by each investigator.

7. Following incubation with blocking solution, aspirate or flick out well contents fromantibody-coated plate, then wash plate four times as in step 3.

8. Add 100 µl of each dilution of IL-18 reference standard and unknown sample toduplicate wells of the antibody-coated EIA plate. Add 100 µl assay buffer to at leasttwo wells of the EIA plate for determination of background. Incubate for 2 hr at roomtemperature (20° to 25°C).

Detect captured IL-18 using peroxidase-conjugated antibody9. Aspirate or flick out well contents from plate, then wash plate four times as in step

3.

10. Dilute peroxidase-conjugated 159-12B (anti-human IL-18 monoclonal antibody) orperoxidase-conjugated 93-10C (anti-mouse IL-18 monoclonal antibody) to 0.5 µg/mlin conjugation buffer. Add 100 µl of the diluted antibody to each well and incubatefor 2 hr at room temperature.

11. Wash plate four times as in step 3, then add 100 µl chromogenic substrate for detectingperoxidase to each well. Incubate for 30 min at room temperature.

To avoid drying up of the microtiter wells, the substrate reagent must be dispensed into thewells as soon as the washing buffer is removed.

12. Add 100 µl stop solution to each well, then read plates in ELISA plate reader atappropriate wavelength for peroxidase substrate employed within 30 min afterstopping reaction.

13. Construct a standard curve (absorbance versus pg/ml IL-18) and calculate theconcentration of IL-18 in the unknown samples using this standard curve.

See UNIT 6.14 for calculation of results in cytokine ELISA.

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Cytokines andTheir CellularReceptors

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocl steps. For common stock solutions, see APPENDIX2A; for suppliers, see APPENDIX 5.

Assay bufferDulbecco’s phosphate-buffered saline (D-PBS; e.g., Life Technologies) contain-

ing:1% (w/v) bovine serum albumin (BSA)5% (v/v) fetal bovine serum (FBS)1 M NaClStore up to 4 weeks at 4°C

ELISA coating antibodyFor human IL-18 assay: Use mouse anti-human IL-18 monoclonal antibody (clone125-2H).For mouse IL-18 assay: Use rat anti-mouse IL-18 monoclonal antibody (clone 74).

Both monoclonal antibodies are commercially available from MBL International.

Conjugation bufferDulbecco’s phosphate-buffered saline (D-PBS; e.g., Life Technologies) contain-

ing:1% (w/v) bovine serum albumin (BSA)5% (v/v) FBS0.1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate

(CHAPS)0.3 M NaClStore up to 2 weeks at 4°C

RPMI-10 medium, completePrepare RPMI 1640 medium supplemented with: 10% (v/v) fetal bovine serum (FBS, Hyclone)50 µM 2-mercaptoethanol (2-ME)2 mM L-glutamine100 U/ml penicillin100 µg/ml streptomycin1 mM sodium pyruvateStore up to 2 months at 4°C

COMMENTARY

Background InformationIL-18 was originally identified (Okamura et

al., 1995) as a potent IFN-γ-inducing factor(IGIF) in the sera and the livers of mice that hadbeen sequentially administered Propionibac-terium acnes and lipopolysaccharide (LPS).The resulting purified material from the ex-tracts of liver tissues from P. acnes–primed andLPS-challenged mice is a single protein withstrong activity inducing IFN-γ production fromanti-CD3-stimulated T cells, particularly in thepresence of IL-12. This homogeneously puri-fied protein was designated as IGIF (Okamuraet al., 1995).

IGIF is a single peptide chain with molecularweight of 18,000 and pI of 4.8, and shows noidentity with any other reported proteins (Oka-mura et al., 1995). Cloned murine and humanIL-18 cDNA (i.e., the proform) encode thenovel proteins consisting of 192 and 193 aminoacids, respectively (Okamura et al., 1995;Ushio et al., 1996). The predicted human IL-18amino acid sequence is 65% homologous overthat of the murine IL-18. From these predictedamino acid sequences, it became clear thatIL-18 lacks usual leader sequence necessary forthe secretion of the mature form of IL-18 acrossthe cell membrane, but instead contains anunusual leader sequence consisting of 35 amino

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Measurement ofHuman and

MouseInterleukin-18

acids at its N-terminus. Like IL-1β, IL-18 issynthesized as a 24-kDa precursor protein,which is then enzymatically cleaved into an18-kDa biologically active mature protein bythe action of the intracellular cysteine protei-nase, IL-1β converting enzyme (also called ICEor caspase-1; Gu et al., 1997). In addition,proteinase 3, a serine protease stored in thegranules of neutrophils and monocytes, hasbeen reported to be an alternative extracellularprocessing enzyme for IL-1β and IL-18 (Fan-tuzzi and Dinarello, 1999). Finally, we havedemonstrated that mature IL-18 is secretedfrom macrophages upon stimulation with FasLindependently of caspase-1, as it is in the caseof 1L-1β from neutrophils (Tsutsui et al.,1999). These results indicated that productionof mature IL-18 is strictly regulated in severaldistinct levels.

Producing cellsIL-18 is produced not only by various types

of immune-competent cells but also nonim-mune cells. Murine macrophages, such asKupffer cells, splenic macrophages, alveolarmacrophages, peritoneal exudate cells, and mi-croglia secrete mature functional IL-18 onlyafter activation with appropriate stimuli(Nakanishi et al., 2001a,b). In contrast, humanperipheral blood mononuclear cells (PBMC)can secrete pro-IL-18 under normal condition(Puren et al., 1999), suggesting that humanpro-IL-18 from PBMC may undergo a changeinto mature IL-18 extracellularly (Fantuzzi andDinarello, 1999).

Murine dendritic cells, either freshly pre-pared or derived by culture of bone marrowcells with IL-4 and GM-CSF, express IL-18mRNA. Human dendritic cells derived fromhuman PBMC constitutively express IL-18mRNA and produce mature IL-18 (Stoll et al.,1998). Epidermal cells, particularly keratino-cytes, can secrete IL-18 with IL-12, in responseto the stimulation with contact allergen (Stollet al., 1997). However, keratinocytes do nothave caspase-1 under normal conditions, sug-gesting that IL-18 secretion by keratinocytesmight be induced in a caspase-1-independentmanner, presumably by extracellular protei-nase 3.

IL-18 also has been reported to be expressedin other non-immune cells (Nakanishi et al.,2001b). These include intestinal epithelialcells, airway epithelial cells, and osteoblasticstromal cells. The latter produce IL-18, whichinhibits osteoclast-like cell differentiation viainduction of GM-CSF production in lympho-

cytes, suggesting that IL-18 may regulate bonemetabolism. IL-18 mRNA is induced in theadrenal cortex, particularly in the zona reticu-laris and zona fasciculata, which contain cellsthat produce glucocorticoid following acutecold stress. Finally, IL-18 secretion is detectedin the central nervous system. These data allowus to hypothesize that IL-18, like IL-1, mayplay an important role in connecting the im-mune system to the endocrine and nervoussystems.

Biological functionStudies performed in vitro has shown that

IL-18 induces IFN-γ production by lympho-cytes, such as T cells, B cells, and NK cells,particularly in the presence of IL-12 (Okamuraet al., 1995; Yoshimoto et al., 1997; Tsutsui etal., 1997; Yoshimoto et al., 1998; Okamura etal., 1998; Dinarello, 1999; Tominaga et al.,2000; Akira, 2000). Furthermore, IL-18 acts asa co-stimulant for Th1 cells to augment IFN-γ,IL-2, GM-CSF, and IL-2Rα production, andinduces cell proliferation, whereas IL-18 hasno effect on Th2 clones (Robinson et al., 97;Kohno et al., 1997; Yoshimoto et al., 1998).Indeed, only Th1 cells express IL-18R (Yoshi-moto et al., 1998), which is composed of IL-18Rα (ligand-binding subunit of IL-18R) andIL-18Rβ (signaling molecule) (O’Neill and Di-narello, 2000; Akira, 2000; Nakanishi et al.,2001a,b). NK cells constutively express IL-18R and IL-18 augments NK activity via thisreceptor. (Hyodo et al., 1999). In addition, IL-18 up-regulates FasL-mediated cytotoxic activ-ity of cloned Th1 cells and NK cells (Dao et al.,1996; Tsutsui et al., 1996).

Cytokine-inducing activity of IL-18 hasbeen mainly investigated using cells stimulatedby IL-12, because IL-12 must first induce IL-18Rα and IL-18 acts on only IL-18Rα-express-ing cells to produce IFN-γ. However, recentstudies by the authors of this unit have shownthat naive T cells and NK cells express low andsubstantial levels of IL-18Rα, respectively(Yoshimoto et al., 1998; Hyodo et al., 1999).Others have shown that IL-18 stimulation ofthese cells leads to production of IL-13 andGM-CSF, particularly in collaboration with IL-2 stimulation (Hoshino et al., 1999). In addi-tion, we have demonstrated that naive CD4+ Tcells cultured with IL-2 and IL-18 without TCRengagement for 4 days express CD40L andproduce modest amounts of IL-4 and a largeamount of IL-13 (Yoshimoto et al., 2000). Fi-nally, we have shown that additional stimula-tion with immobilized anti-CD3 augments

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Cytokines andTheir CellularReceptors

their capacity to produce these Th2 cytokinesand to develop into Th2 cells. Taken togehter,these studies show that IL-18 has the potentialto induce Th2 cell in an IL-4-dependent manner(Yoshimoto et al., 2000).

In a related vein, we have demonstrated thatbasophils and mast cells derived by culture ofbone marrow cells with IL-3 for 10 days expressIL-18Rα and that basophils produce largeamounts of IL-4 and IL-13 in response to stimu-lation with IL-3 and IL-18, although theirpathophysiological relevance is still to be elu-cidated (Yoshimoto et al., 1999). As mast cellsand basophils are major inducers and effectorsof allergic inflammation, these data suggeststhat IL-18 might be critically involved in induc-tion of allergic inflammation.

Critical Parameters

BioassaysIL-18 ELISA is the most accurate method

to measure the amount of IL-18. However,ELISA for IL-18 detects not only biologicallyactive mature IL-18 but also biologically inac-tive precursor IL-18 (Taniguchi et al., 1997;Kikkawa et al., 2000). To identify the biologicalactivity of IL-18, we need to perform a bioassayfor IL-18 as shown in Basic Protocol 1. Thesebioassays are based on the ability of IL-18 toinduce IL-12-stimulated T cells to secrete IFN-γ. Thus, mouse splenic T cells stimulated withIL-12 for 72 hr express both high- and low-af-finity IL-18R, and show dose-dependent IFN-γproduction in response to IL-18 (Yoshimoto etal., 1998). Human T cells stimulated with IL-12for 6 days are also competent to respond toIL-18 with IFN-γ production (Tominaga et al.,2000). IL-12-stimulated human T cells can re-spond to human IL-1β to induce IFN-γ produc-tion (Tominaga et al., 2000) whenever IL-12-stimulated mouse T cells fail to respond tomouse IL-1β. Thus, the human assay mustincorporate anti-IL-1β to neutralize any of thelatter present in unknown samples. Activitydetected in these bioassays can be shown to bedue to IL-18 by addition of neutralizing anti-IL-18 antibody such as clone 125-2H (for hu-man IL-18) or clone 93-10C (for mouse IL-18).

With IL-12, IL-18 also induces mouse andhuman T cells to proliferate (Yoshimoto et al.,1998; Tominaga et al., 2000). Thus, a lympho-cyte proliferation assay is an alternate methodfor the measurement of IL-18. Using mouse Tcells, the dose of rIL-18 that produces 50% ofthe maximal T cell proliferation caused by asaturating amount of IL-18 is the same as that

obtained by T cell IFN-γ production. However,using human T cells, this bioassay is somewhatmore sensitive than the assay of T cell IFN-γproduction. Additional reagents and equipmentfor using proliferation as a read-out are neces-sary for this alternative method.

While the standard assay for human andmouse IL-18 using IL-12-stimulated T cells issensitive and useful, the preparation of T cellsfor use in the assay is tedious. Cloned murineNK cells (LNK5E3 cell) can respond to mouseIL-18 to induce IFN-γ production (Tsutsui etal., 1996; Tsutsui et al., 1999). While an assaybased on these cells in simpler to perform thanthe standard assay using IL-12-pretreated Tcells, it may give rise to spurious results becauseother factors, such as IL-15, IL-2, and IL-12 inthe unknown samples, may synergistically in-duce IFN-γ production from NK cells. It hasalso been established that KG-1 cell (humanmyelomonocyte; ATCC no. CCL246; Konishiet al., 1997) and mouse IL-18 receptor–trans-fected KG-1 cell (Taniguchi et al., 1998) canproduce IFN-γ in response to human or mouseIL-18, respectively; these cells may also proveuseful for the bioassay of IL-18.

ELISATaniguchi and co-workers have established

and characterized 13 anti-human IL-18 mono-clonal antibodies (7 mouse hybridomas and 6rat hybridomas; Taniguchi et al., 1997). In west-ern blot analysis, three murine monoclonal an-tibodies showed strong reactivity with mem-brane-blotted human IL-18, while all six ratmonoclonal antibodies showed no reactivity inthe same assay. In human IL-18 bioassay, onemouse monoclonal antibody (clone 125-2H)and six rat monoclonal antibodies neutralizedIL-18. Neutralizing activities of the six ratmonoclonal antibodies were 10-fold lower thanthat of clone 125-2H. A concentration of 1µg/ml of 125-2H completely inhibits IFN-γproduction induced by human IL-18 (40 ng/ml)in KG-1 cell (Konishi et al., 1997). The ELISAcomposed of two monoclonal antibodies (125-2H as the capture reagent and rat monoclonalantibody 159-12B as the detector antibody) isthe most sensitive, and correlated well with theresults of human IL-18 bioassay. This ELISAdetects human IL-18 with a minimum detectionlimit of 12.5 pg/ml, but does not react withheat-denatured human IL-18 or functionallyinactive IL-18. The ELISA does not show anycross-react ivi ty with other cytokines(Taniguchi et al., 1997). Several monoclonalantibodies against human IL-18 are commer-

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1001010.10.01

without anti-IL-18 Ab

with anti-IL-18 Ab

Mouse IL-18 (ng/ml)

IFN

-γ (n

g/m

l)

80

60

40

20

0

Figure 6.26.1 IL-18 and IL-12 synergize for IFN-γ production from mouse T cells. Mouse splenicT cells (2 × 105/0.2 ml/well) were cultured with IL-12 (20 ng/ml) plus IL-18 (0 to 100 ng/ml) in thepresence (open circles) or absence (closed circles) of anti-IL-18 Ab (10 µg/ml) in 96-well plates for72 hr. Culture supernatants were harvested and tested for mouse IFN-γ production by ELISA.Results are mean ± 1 SD of triplicate cultures.

1007550250

without anti-IL-18 Ab

with anti-IL-18 Ab

Human IL-18 (ng/ml)

IFN

-γ (n

g/m

l)

15

10

5

0

Figure 6.26.2 IL-18 and IL-12 synergize for IFN-γ production from human T cells. Human T cells(2 × 105/0.2 ml/well) were cultured with IL-12 (100 ng/ml) plus IL-18 (0 to 100 ng/ml) in the presence(open circles) or absence (closed circles) of anti-IL-18R Ab (10 µg/ml) in 96-well plates for 6 days.Culture supernatants were harvested and tested for human IFN-γ production by ELISA. Results aremean ± 1 SD of triplicate cultures.

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cially available; however some of them mayreact with inactive precursor IL-18 (Kikkawaet al., 2000). So far, ELISA using two neutral-izing monoclonal antibodies, 125-2H and 159-12B, is most sensitive to biological active hu-man IL-18. Mouse IL-18 ELISA, comprisingtwo monoclonal antibodies (clone 74 as thecapture reagent and clone 93-10C as the detec-tor antibody) is sensitive, and is the most accu-rate method to measure the amount of mouseIL-18. A concentration of 0.5 µg/ml 93-10Cmoderately (>50%) inhibits, and 5 µg/ml of thisantibody completely (>90%) inhibits IFN-γproduction induced by mouse IL-18 (30 ng/ml)in mouse IL-18 receptor–transfected KG-1cells (Taniguchi et al., 1998). However thismouse IL-18 ELISA may react with pro-IL-18.Thus, further biological functional assay todetect mature active mouse IL-18 is necessary.

Anticipated ResultsResults of a representative IFN-γ production

by IL-12-stimulated mouse or human T cells inresponse to mouse or human IL-18 are shownin Figure 6.26.1 and Figure 6.26.2, respectively.T cells show dose-dependent IFN-γ productionin response to IL-18 and IL-12. Anti-mouseIL-18 antibody (10 µg/ml) can completelyblock mouse IL-18–induced IFN-γ production(Fig. 6.26.1). Anti-human IL-18R monoclonalantibody (clone 117-10C; 10 µg/ml) can com-

pletely block human IL-18-induced IFN-γ pro-duction (Figure 6.26.2).

Representative standard curves for theELISA are shown in Figure 6.26.3. The sensi-tivity of the ELISA assay is 12.5 pg/ml (humanIL-18) and 25.0 pg/ml (mouse IL-18). Serumsamples from healthy volunteers contain 126.0± 45 pg/ml of IL-18 assayed by the humanIL-18 ELISA.

Literature CitedAkira, S. 2000. The role of IL-18 in innate immunity.

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Dinarello, C. A. 1999. IL-18: A TH1-inducing,proinflammatory cytokine and new member ofthe IL-1 family. J. Allergy Clin. Immunol.103:11-24.

Dao, T., Ohashi, K., Kayano, T., Kurimoto, M., andOkamura H. 1996. Interferon-γ-inducing factor,a novel cytokine, enhances Fas ligand- mediatedcytotoxicity of murine T helper 1 cells. CellImmunol. 173:230-235.

Fantuzzi, G. and Dinarello, C.A. 1999. Interleukin-18 and interleukin-1β: Two cytokine substratesfor ICE (caspase-1) J. Clin. Immunology 19:1-11.

Fujimori, Y., Takatsuka, H., Takemoto, Y., Hara, H.Okamura, H., Nakanishi, K., and Kakishita, E.2000. Elevated interleukin (IL)-18 levels duringacute graft-versus-host disease after allogenicbone marrow transplantation. Br. J. Haematol.109: 652-657.

human IL-18

mouse IL-18

100 1000100

0.5

1.0

1.5

2.0

2.5

IL-18 (pg/ml)

Abs

orba

nce

(450

nm

)

Figure 6.26.3 Representative results of human and mouse IL-18 ELISAs. EIA plates coated withthe indicated antibody against human or mouse IL-18 were used to assay human or mouse IL-18,respectively.

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Gu, Y., Kuida, K., Tsutsui, H., Ku, G., Hsiao, K.,Fleming, M.A.. Hayashi, N., Higashino, K.,Okamura, H., Nakanishi, K., Kurimoto, M.,Tanimoto, T., Flavell, R.A., Sato, V., Harding,M.W., Livingston, D.J., and Su, M.S. 1997. Ac-tivation of interferon-γ inducing factor mediatedby interleukin-1β converting enzyme. Science275:206-209.

Hoshino, T., Wiltrout, R.H., and Young, H.A. 1999.IL-18 is a potent coinducer of IL-13 in NK andT cells: A new potential role for IL-18 in modu-lating the immune response. J. Immunol.162:5070-5077.

Hoshino, T., Yagita, H., Ortaldo, J.R., Wiltrout, R.H.,and Young H.A. 2000. In vivo administration ofIL-18 can induce IgE production through Th2cytokine induction and up-regulation of CD40ligand (CD154) expression on CD4+ T cells. Eur.J. Immunol. 30:1998-2006.

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Kikkawa, S., Shida, K., Okamura, H., Begum, N.A.,Matsumoto, M., Tsuji, S., Nomura, M., Suzuki,Y., Toyoshima, K., and Seya, T. 2000. A com-parative analysis of the antigenic, structural, andfunctional properties of three different prepara-tions of recombinant human interleukin-18. J.Interferon Cytokine Res. 20:179-85.

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Kohno, K., Kataoka, J., Ohtsuki, T., Suremoto, Y.Okamoto, I., Usui, M., Ikeda, M., and Kurimoto,M. 1997. IFN-γ-inducing factor (IGIF) is acostimulatory factor on the activation of Th1 butnot Th2 cells and exerts its effect independentlyof IL-12. J. Immunol. 158:1541-1550.

Nakamura, H., Komatsu, K., Ayaki, M., Kawamoto,S., Murakami, M., Uoshima, N., Yagi, T.,Hasegawa, T., Yasumi, M., Karasumoto, T.,Teshima, H., Hiraoka, A., and Masaoka, T. 2000.Serum levels of soluble IL-2 receptor, and IL-12,IL-18, and IFN-γ in patients with acute graft-ver-sus-host disease after allogenic bone marrowtransplantation. J. Allergy Clin. Immunol.106:45-50.

Nakanishi, K., Yoshimoto, T., Tsutsui, H., and Oka-mura, H. 2001a. Interleukin-18 is a unique cy-tokine that stimulates both Th1 and Th2 re-sponses depending on its cytokine milieu. Cytok-ine Growth Factor Rev. 12:53-72.

Nakanishi, K., Yoshimoto, T., Tsutsui, H., and Oka-mura, H. 2001b. Interleukin-18 regulates bothTh1 and Th2 responses. Annu. Rev. Immunol.19:423-74.

Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M.,Hakura, A., Tanimoto, T., Torigoe, K., Okura, T.,Nukada, Y., Hattori, K., Akita, K., Namba, M.,Tanabe, F., Konishi, K., Fukuda, S., and Kuri-moto, M. 1995. Cloning of a new cytokine thatinduces IFN-γ production by T cells. Nature378:88-91.

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Okamura, H., Kashiwamura, S.-I., Tsutsui, H.,Yoshimoto, T., and Nakanishi, K. 1998. Regula-tion of interferon-gamma (IFN-γ) production byIL-12 and IL-18. Curr. Opin. Immunol. 10:259-264.

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Puren, A.J., Fantuzzi, G., and Dinarello, C.A. 1999.Gene expression, synthesis, and secretion of in-terleukin 18 and interleukin 1β are differentiallyregulated in human blood mononuclear cells andmouse spleen cells. Proc. Natl. Acad. Sci. U.S.A.96:2256-2261.

Robinson, D., Shibuya, K., Mui, A., Zonin, F. Mur-phy, E., Sana, T., Hartley, S.B., Menon, S., Kas-telein, R., Bazan, F., and O’Garra, A. 1997. IGIFdoes not drive Th1 development but synergizeswith IL-12 for interferon-γ production and acti-vates IRAK and NF-κB. Immunity 7:571-581.

Stoll, S., Muller, G., Kurimoto, M., Saloga, J. Tani-moto, T., Yamauchi, H., Okamura, H., Knop, J.,and Enk, A. H. 1997. Production of IL-18 (IFN-γ-inducing factor) messenger RNA and func-tional protein by murine keratinocytes. J. Immu-nol. 159:298-302.

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Taniguchi, M., Nagaoka, K., Ushio, S., Nukada, Y.,Okura, T., Mori, T., Yamauchi, H., Ohta, T.,Ikegami, H., and Kurimoto, M. 1998. Estab-lishment of the cells useful for murine inter-leukin-18 bioassay by introducing murine inter-leukin-18 receptor cDNA into human myelo-monocytic KG-1 cells. J. Immunol. Methods.217:97-102.

Tominaga, K., Yoshimoto, T., Torigoe, K., Kuri-moto, M., Matsui, K., Hada, T., Okamura, H.,and Nakanishi, K. 2000. IL-12 synergizes withIL-18 or IL-1β for IFN-γ production from humanT cells. Int. Immunol. 12:151-160.

Tsutsui, H., Nakanishi, K., Matsui, K., Higashino,K., Okamura, H., Miyazawa, Y., and Kaneda, K.1996. Interferon-γ-inducing factor up-regulatesFas ligand-mediated cytotoxic activity of murinenatural killer cell clones. J. Immunol. 157:3967-3973.

Tsutsui, H., Matsui, K., Kawada, N., Hyodo, Y.Hayashi, N., Okamura, H., Higashino, K., andNakanishi, K. 1997. IL-18 accounts for bothTNF-α- and Fas ligand-mediated hepatotoxicpathways in endotoxin-induced liver injury inmice. J. Immunol. 159:3961-3967.

Tsutsui, H., Kayagaki, N., Kuida, K., Nakano, H.Hayashi, N., Takeda, K., Matsui, K., Kashiwa-mura, S., Hada, T., Akira, S., Yagita, H., Oka-mura, H., and Nakanishi, K. 1999. Caspase-1-in-dependent, Fas/Fas ligand-mediated IL-18 se-cretion from macrophages causes acute liverinjury in mice. Immunity 11:359-367.

Ushio, S., Namba, M., Okura, T., Hattori, K.Nukada, Y., Akita, K., Tanabe, F., Konishi, K.,Micallef, M., Fujii, M., Torigoe, K., Tanimoto,T., Fukuda, S., Ikeda, M., Okamura, H., andKurimoto, M. 1996. Cloning of the cDNA forhuman IFN-γ-inducing factor, expression in Es-cherichia coli, and studies on the biologic activi-ties of the protein. J. Immunol. 156:4274-4279.

Yoshimoto, T., Okamura, H., Tagawa, Y.-I., Iwakura,Y., and Nakanishi K. 1997. Interleukin 18 to-gether with interleukin 12 inhibits IgE produc-tion by induction of interferon-γ production fromactivated B cells. Proc. Natl. Acad. Sci. U.S.A.94:3948-3953.

Yoshimoto, T., Takeda, K., Tanaka, T., Ohkusu, K.,Okamura, H., Akira, S., and Nakanishi, K. 1998.IL-12 up-regulates IL-18 receptor expression onT cells, Th1 cells, and B cells: synergism withIL-18 for IFN-γ production. J. Immunol.161:3400-3407.

Yoshimoto, T., Tsutsui, H., Tominaga, K., Hoshino,K., Okamura, H., Akira, S., Paul, W.E., andNakanishi, K. 1999. IL-18, although anti-aller-gic when administered with IL-12, stimulatesIL-4 and histamine release by basophils. Proc.Natl. Acad. Sci. U.S.A. 96:13962-13966.

Yoshimoto, T., Mizutani, H., Tsutsui, H., Noben-Trauth, N., Yamanaka, K-I., Tanaka, M., Izumi,S., Okamura, H., Paul, W.E., and Nakanishi, K.2000. IL-18 induction of IgE: Dependence onCD4+ T cells, IL-4 and Stat6. Nature Immunol.1:132-137.

Key ReferenceOkamura, H. et al. 1995. See above.

An original paper of IL-18.

Nakanishi, K. et al., 2001b. See above.

Excellent reviews of all facts of IL-18 biology, bio-chemistry, and molecular biology.

Contributed by Tomohiro Yoshimoto, Hiroko Tsutsui, Haruki Okamura, and Kenji NakanishiHyogo College of MedicineHyogo, Japan

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