IL18 Chemoattraction of Human T Cells by

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Chemoattraction of Human T Cells by IL-18

Damo Xu, Neil Thomson, Iain B. McInnes and Foo Y. LiewMousa Komai-Koma, J. Alastair Gracie, Xiao-qing Wei,

http://www.jimmunol.org/content/170/2/10842003; 170:1084-1090; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Chemoattraction of Human T Cells by IL-181

Mousa Komai-Koma, J. Alastair Gracie, Xiao-qing Wei, Damo Xu, Neil Thomson,Iain B. McInnes, and Foo Y. Liew2

Cell locomotion is crucial to the induction of an effective immune response. We report here the chemoattraction of CD4� T cellsby IL-18, a member of the IL-1 cytokine family. Recombinant IL-18 increased the proportion of T cells in polarized morphologyin vitro and stimulated their subsequent invasion into collagen gels in an IL-18 concentration gradient-dependent manner. Im-munofluorescent microscopy studies determined that the major cell type responding to IL-18 was IL-18R�CD4�. Importantly,synovial CD4� T cells from patients with rheumatoid arthritis responded to IL-18, adopting polarized morphology and gelinvasion without further activation ex vivo, indicating the physiologic relevance of our observations. Finally, injection of rIL-18into the footpad of DBA/1 mice led to local accumulation of inflammatory cells. These data therefore demonstrate for the first timelymphocyte chemoattractant properties of a member of the IL-1 cytokine family and its relevance in inflammatory diseases. TheJournal of Immunology, 2003, 170: 1084–1090.

I nterleukin 18, a member of the IL-1 cytokine family, medi-ates important activities during both acquired and innate im-mune responses. Initially characterized by its capacity to pro-

mote Th1 responses in synergy with IL-12 (1, 2), IL-18 hasrecently been shown to drive either Th1 or Th2 responses, depen-dent on the cytokine microenvironment, suggesting a broader rolein functional T cell differentiation than that originally recognized(3, 4). Sustained IL-18R� expression on Th1 cells however sug-gests preferential activation of the latter in chronic lesions, e.g., inrheumatoid arthritis synovitis (5). A role in innate responses is alsoproposed. IL-18 enhances NK cell cytotoxicity and directly in-duces IFN-� production by NK cells (6). Moreover, we recentlydemonstrated an important role for IL-18 in activating neutrophilsand in promoting their recruitment to the peritoneal cavity in vivo(7). IL-18 can also directly induce monokine production by mac-rophages that constitutively express IL-18R� (5).

IL-18 has been implicated as a critical factor in host responsesin a broad range of infectious and autoimmune diseases. ProtectiveTh1 responses during various bacterial and viral infections may beabrogated or enhanced by manipulation of IL-18 expression inIL-18-intact mice (8, 9). IL-18-deficient mice exhibit altered re-sponsiveness to Mycobacterium bovis, Propionibacterium acnes,and Leishmania major, associated with modified T cell/NK cellfunction (6, 10). IL-18 mRNA is up-regulated in nonobese diabeticmice and the murine IL-18 gene maps to the idd2 susceptibilitylocus (11). Similarly, IL-18-deficiency is associated with alteredmyelin oligodendrocytic glycoprotein peptide-specific autoreac-tive T cell responses and amelioration of autoimmune encephalo-myelitis (12). Several compelling data now implicate IL-18 in thepathogenesis of human inflammatory disease states including rheu-

matoid arthritis (RA),3 psoriasis, sarcoidosis, adult onset Still’sdisease, and inflammatory bowel disease (13–16). In particular, wehave recently proposed that IL-18 exerts important proinflamma-tory activity in RA synovial tissues (17). IL-18-deficient mice de-velop significantly reduced incidence and severity of collagen-in-duced arthritis compared with wild-type mice (18) and Ab-, orIL-18-binding protein, mediated IL-18 neutralization suppressesstreptococcal cell wall-induced arthritis and collagen-induced ar-thritis (19, 20). The mechanisms whereby IL-18 mediates effects insynovium remain unclear, but likely include modulating articularTh1 cell responses, directly promoting macrophage TNF-� produc-tion (5) and enhancing endothelial activation and angiogenesis (21).

We have explored the hypothesis that IL-18 can contribute toacquired immune response development and to the maintenance ofchronic immune stimulation in diseases such as RA by promotingchemotaxis of activated T cells. The present report documents forthe first time that IL-18 induces human CD4� lymphocyte che-motaxis. IL-18 induced T cell polarization and migration into col-lagen gel matrices. Moreover, we demonstrated that such activityresides primarily in human Th1 cells that retain high levels ofIL-18R� expression. RA synovial CD4�, but not CD8� T cellsalso migrate to IL-18, suggesting that our observations have phys-iologic relevance. Finally, we have shown that IL-18 can inducelocal mononuclear cell recruitment in vivo. Together these datashow that IL-18 is chemotactic for T lymphocytes and as suchprovide a novel mechanism whereby IL-18 can promote and sus-tain inflammatory responses.

Materials and MethodsReagents

PE-conjugated anti-human IL-18R Ab and anti-human IL-18 Ab were ob-tained from R & D Systems (Abingdon, U.K.). Recombinant human IL-18was cloned, expressed, and purified as described previously (5). Murinerecombinant IL-18 was obtained from PeproTech (London, U.K.). Cyto-kines used were free of LPS as assessed by the Limulus amebocyte assay(Sigma-Aldrich, Poole, U.K.). FITC-conjugated anti-human CD4, CD8,CD19, CD14, CD56, and PE- and FITC-conjugated isotype control mouseIgG1 were obtained from Sigma-Aldrich. The medium routinely used wasRPMI 1640 supplemented with 25 �M 2-ME (Sigma-Aldrich), penicillin

Division of Immunology, Infection and Inflammation, Faculty of Medicine, Univer-sity of Glasgow, Glasgow, United Kingdom

Received for publication July 12, 2002. Accepted for publication November 8, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Wellcome Trust, Medical Research Council, theArthritis Research Campaign, and the Chief Scientist’s Office, Scotland.2 Address correspondence and reprint requests to Dr. Foo Y. Liew, Department ofImmunology and Bacteriology, Western Infirmary, University of Glasgow, GlasgowG11 6NT, U.K. E-mail address: f.y.liew@clinmed.gla.ac.uk

3 Abbreviations used in this paper: RA, rheumatoid arthritis; SEB, staphylococcalenterotoxin B.

The Journal of Immunology

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(100 IU/ml), streptomycin (100 �g/ml), L-glutamine (2 mM), and FCS(10%) (all obtained from Life Technologies, Paisley, U.K.).

Cell preparation and culture.

Heparinized venous blood was withdrawn gently from the forearm ofhealthy donors. PBMCs were purified by standard procedures using densitygradient separation medium (Lymphoprep; Nycom Pharma, Oslo, Nor-way). The cells were washed extensively and either resuspended in RPMI1640–10% FCS and used immediately for polarization assay or culturedovernight at 37°C in 5% CO2 in 1 ml of medium (2 � 106 cells/ml) in24-well dishes in the presence or absence of staphylococcal enterotoxin B(SEB, 1 �g/ml; Sigma-Aldrich) as described previously (22). After wash-ing, most of the cells reverted from the locomotive to spherical morphol-ogy. Human umbilical cord blood was obtained during full-term cesareandeliveries under appropriate ethical consent (North Glasgow Trust EthicalCommittee, Glasgow, U.K.). CD4� T cell populations were negativelypurified from human umbilical cord blood by high-gradient magnetic sort-ing using the Viro MACS magnetic columns (Miltenyi Biotec, Auburn,CA) according to the manufacturer’s recommendations. The depletion mix-ture consisted of hapten-conjugated Abs (CD8, CD11b, CD16, CD19,CD36, and CD56). Purity of CD4� T cells was �97% as judged by flowcytometry analysis using anti-CD14, CD3, CD19, CD4, CD56, and CD8mAbs. To prepare Th1 cell populations, freshly purified CD4� T cells werecultured in 25-ml tissue culture flasks (Nunc, Roskilde, Denmark) at 0.5 �106 cells/ml in the presence of PHA (1 �g/ml; Sigma-Aldrich), rIL-2 (100ng/ml; R & D Systems), rIL-12 (2 ng/ml; BD PharMingen, San Diego,CA), and neutralizing anti-IL-4 Ab (200 ng/ml; BD PharMingen). Threedays later, IL-2 (10 ng/ml) was added and the cells were harvested on day5 for analysis. Synovial fluid was obtained from six patients with RAsatisfying the American College of Rheumatology criteria (23). Mononu-clear cells were separated as described above. The cells were used imme-diately after separation.

Flow cytometry

Surface markers of the cells were analyzed by FACSCalibur flow cytom-etry using CellQuest software (BD Biosciences, Mountain View, CA). Toprevent nonspecific binding, all samples were preincubated for 30 min withhuman IgG (Sigma-Aldrich) to block the FcR. Aliquots of fresh cells orcultured cells (3 � 105 cells/tube) were suspended in buffer containing 2%FCS and 0.02% sodium azide in PBS (staining buffer) and stained for20–30 min with specific FITC- or PE-conjugated mAbs. Cells labeled withFITC- or PE-conjugated mouse isotype-matched Abs were used ascontrols.

Immunofluorescence microscopy

This allows a direct phenotype visualization of polarized cells. The cellswere fixed with 1% paraformaldehyde (Sigma-Aldrich) for 10–15 min toretain locomotor morphology, then attached to poly-L-lysine-coated cov-erslips (2 mg/ml; Sigma-Aldrich), and stained with appropriate FITC- orPE-labeled markers. Marker-positive and -negative, polarized and spheri-cal cells were counted using a �40 oil phase-contrast objective of a ZeissAxioskop fluorescence microscope (Zeiss, Oberkochen, Germany).

Cytospin staining

Cell morphology using Giemsa-stained preparations is a useful preliminaryto phenotype synovial cells (n � 6 patients). The morphology of these cellswas observed under the optical microscope: neutrophils (77.50 � 1.50%),lymphocytes (17.33 � 1.23%), and monocytes (5.17 � 0.6%).

Polarization assay

This assay measures the change from a spherical shape to the shape changecharacterized by head-tail polarity, typical of locomotive cells (24). Freshlyisolated or overnight-cultured PBMCs were resuspended in RPMI 1640medium supplemented with 10% FCS and mixed with human IL-18 orIL-15 at concentrations ranging from 0.1 to 1 �g/ml. Experiments wereconducted using polystyrene round-bottom tubes (110 � 16 mm; Sterilin,Stone, U.K.). The tubes, containing 200 �l of cell suspension at a concen-tration of 2 � 106/ml, were incubated for 30 min at 37°C. A basal controlvalue was established using RPMI 1640-FCS alone. The cells were fixedusing 200 �l of 2.5% glutaraldehyde (Sigma-Aldrich) and the proportionsof cells showing head-tail polarization typical of locomotive cells weredetermined. The proportion of cells scored as either spherical (nonmotile)or polarized (motile) was counted directly using a �40 phase-contrast ob-jective. Cells (250–300) were counted blind, and polarized cells were ex-pressed as a percentage of viable cells. Data presented are values aftersubtracting the background values (without cytokine), which ranged from

5 � 0.8% for freshly isolated cells, 18 � 1.7% for cells cultured withmedium alone overnight, and 23 � 2.3% for cell cultured with SEB.

Collagen gel invasion assay

Rat tail collagen (type I) was prepared in solution from freshly obtained rattail tendons by established methods (25). Gels were formed by bringingsoluble collagen (1.5–2 mg/ml) in dilute acetic acid solution back to phys-iologic pH and osmolarity. IL-18 or FCS in culture medium was addedbefore gelatinization. The mixture was then transferred into 24-well dishes(Sterilin) and allowed to gel. Cells were prepared and activated with ap-propriate activators as described above. They were washed and overlaid onthe gel in 24-well dishes and incubated at 37°C for 20 h (unless otherwisestated) to allow cells to invade the gel. The proportion of invading cells wasdetermined with an inverted microscope by scoring the number of cellsremaining on top of the gel and the number of cells that had penetrated thegel, counting a minimum of 200 cells. The proportion of locomotor cellswas calculated from the ratio of the numbers of invasive and noninvasivecells. To distinguish between chemotaxis and chemokinesis, IL-18 was alsoadded on top of the collagen gel and also in the gel and incubated for 20 h.

Measurement of IL-5, IFN-�, and IL-18

IL-5 and IFN-� concentrations in culture supernatants were determined byELISA using paired Abs (BD PharMingen) and developed with tetrameth-ylbenzidine peroxidase substrate (Insight Biotechnology, Middlesex, U.K.)according to the manufacturer’s recommendations. Sensitivity of the assaywas 10 pg/ml. IL-18 in synovial fluid was similarly determined usingpaired Abs (Diaclone Research, Besancon, France). Sensitivity of the assaywas 45 pg/ml. IL-18 levels in RA synovial fluids were up to 155.4 � 28.7pg/ml (mean � SEM, n � 6).

IL-18-induced cellular migration in vivo

This was conducted as previously described (26, 27). Briefly, male DBA/1mice (Harlan Olac, Oxon, U.K.) and IL-18-deficient mice of the DBA/1background (10, 18), age 10 wk, were injected i.p. with 500 �g of P. acnesand 7 days later received 500 ng of murine rIL-18 or PBS in the hindfootpad. Footpad swelling was measured at 0, 8, 15, and 24 h after injectionusing a constant pressure dial caliper (Kroeplin, Munich, Germany). Fol-lowing the measurement, the mice were sacrificed and the footpads wereremoved, Formalin fixed, and analyzed histologically by H&E staining. Allmice were kept in the Biological Service facilities at the University ofGlasgow according to the U.K. Home Office guidelines.

Immunohistology of synovial membrane

Synovial membranes (arthoplasty specimens) were obtained from RA pa-tients satisfying the American College of Rheumatology diagnostic criteria(n � 12). Formaldehyde-fixed paraffin sections (3 �m) were stained by astandard streptavidin-HRP protocol using an anti-IL-18 mAb (a kind giftfrom Dr. A. Jackson, Cancer Research U.K., Leeds, U.K.) (5). Product wasvisualized with 3,3�-diaminobenzidine tetrachloride (Sigma-Aldrich) andcounterstained with hematoxylin (Sigma-Aldrich). IL-18 expression wasquantified by two independent observers using light microscopy. For ver-ification of specificity, the anti-IL-18 Ab was replaced by an isotype-matched control (Sigma-Aldrich). Neutralization was performed by prein-cubation of primary anti-IL-18 Ab with either 1 �g of rIL-18 or IL-1� (R& D Systems) before tissue staining.

Statistical analysis

Values are expressed as mean � SEM. Statistical analysis was performedusing Student’s t test. A p � 0.05 was considered to be significant.

ResultsIL-18 induces polarization and migration of peripheral blood Tcells

Lymphocyte shape change (polarization) and migration into col-lagen gels represent useful indicators of chemokinetic activity. Wetherefore determined the proportion of PBMCs exhibiting polar-ization in response to increasing concentrations of IL-18. IL-18induced no reproducible effect on freshly isolated lymphocyte pop-ulations (Fig. 1a) reflecting low levels of spontaneous IL-18R�expression (data not shown). It has been shown that SEB promotedcytokine-mediated lymphocyte polarization (22). We thereforestimulated PBMCs with SEB to enhance cell polarization. After

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overnight culture in SEB, PBMCs polarized to IL-18 in a dose-dependent manner that was similar to the response induced byIL-15, a recognized T cell chemoattractant (23). Maximal polar-ization was seen with 100 ng/ml IL-18; at this dose, �20% of totalPBMCs were polarized (Fig. 1a). Flow cytometric analysis of thePBMC fraction contained 65 � 5.6% CD3� cells (n � 5), 6.8 �0.7% CD19� cells (n � 5), 8.5 � 0.5% CD14� cells (n � 2), and

�96% of the cells within the lymphocyte gate expressed CD45(data not shown). Immunofluorescence microscopy analysisshowed that the polarized cells were predominantly CD3�CD4� Tcells, with few CD8� T cells (�5%) assuming polarized morphol-ogy (data not shown). This is not unexpected since CD4� but notCD8� cells are responsive to SEB (28) Commensurate with this,cells cultured for 16 h in SEB, but not in medium alone, increasedthe expression of IL-18R� on CD4� cells (up to 40% of totalCD4� T cell population in PBMCs measured by flow cytometry,data not shown). The polarized and nonpolarized cells are shownin Fig. 1b. We also investigated the ability of the mononuclearcells from human cord blood (which contained principally naivelymphocytes) to polarize in response to IL-18. Cord blood cellspolarized in a similar manner as PBMCs in response to IL-18following activation by SEB, but did not respond to IL-18 whenthey were not activated with SEB (Fig. 1c). A similar percentageof polarized cells was obtained whether purified CD4� T cells orPBMCs were examined (data not shown). As specificity control,we further showed that neutralizing monoclonal anti-human IL-18Ab abolished the chemotactic effects of IL-18 (Fig. 1d). SEB-ac-tivated lymphocytes polarized within 5 min of IL-18 stimulationand reached maximal levels after 30 min (Fig. 1e). Although mea-surement of shape change is a reliable correlate of chemoattrac-tion/locomotion, the assay does not directly measure locomotion.To formally demonstrate this, we used the collagen gel invasionassay. PBMCs were cultured overnight in SEB, washed to removethe supernatant, and overlaid on a collagen gel previously impreg-nated with IL-18 (or diluent alone) in medium/10% FCS. The per-centage of cells migrating into collagen gels in response to IL-18after 20 h was significantly higher than that in the control gels (Fig.1f). Moreover, IL-12 (at a range of concentrations) did not haveany activity in both polarization and gel invasion assays (data notshown), suggesting that this activity is not a general property ofinnate response monokines. These data therefore demonstrateclearly that IL-18 can rapidly induce not only polarization but alsoinvasive locomotion in PBMC subsets.

IL-18 induces migration of Th1 cells

Since we have previously shown that IL-18R� is preferentiallyexpressed on committed Th1 cells (29), we next explored the hy-pothesis that IL-18 induced locomotion in Th1 cells. CD4� cellspurified from human cord blood were cultured under Th1 differ-entiating conditions. Flow cytometry showed that whereas naivecord blood CD4� T cells expressed a low level of IL-18R�, 90%of committed Th1 cells expressed the receptor (data not shown).Predominant IFN-� (1.1 � 0.1 ng/ml), but not IL-5 (�30 pg/ml),production confirmed functional Th1 differentiation (data notshown). IL-18 induced human Th1 cell migration into collagengels in a dose-dependent manner (Fig. 2a). However, only �30%of the Th1 cells migrated into the gel in response to 200 ng/mlIL-18. This is consistent with earlier reports that lymphocytes mustenter G1 phase for full expression of a locomotor phenotype (30).Cell cycle analysis by flow cytometry showed that 60% of thecommitted Th1 cells were in the G0-G1 phase at the time of themigration assay (data not shown). Time course analysis comparingthe proportion and migration distance of cells into collagen gelscontaining 200 ng/ml IL-18 or medium alone showed increasingresponses up to 18 h (Fig. 2, b and c). To confirm that the migra-tion was not due to random chemokinesis, the gel invasion assaywas performed with or without an IL-18 concentration gradient(Fig. 3d). Maximal Th1 cell migration occurred in a positive IL-18concentration gradient between the top of the gel and inside the gel(chemotactic condition). In contrast, no significant migration was

FIGURE 1. IL-18 induces polarization and migration of human periph-eral blood T cells. a, Freshly isolated PBMCs (E) or PBMCs culturedovernight with SEB or medium alone (no added SEB) were stimulated withgraded concentrations of IL-18 or IL-15 for 30 min and fixed, and the cellshape change was examined under phase-contrast microscopy as describedin Materials and Methods. Data are mean � SEM of percent polarized cellsof four experiments. b, PBMCs were cultured overnight with SEB andactivated with 200 ng/ml IL-18 for 30 min and fixed, and the polarized(filled arrow) and nonpolarized (open arrow) cells were scored underphase-contrast microscopy. c, Mononuclear cells from human cord bloodcultured overnight with SEB or freshly isolated were also tested for po-larization in response to IL-18. d, IL-18-induced polarization of SEB-cul-tured PBMCs can be blocked by anti-IL-18 Ab. e, Polarization of SEB-cultured PBMCs was evident 5 min after IL-18 (500 ng/ml) stimulation andpeaked at 30 min. d and e, The background value was not subtracted. f,SEB-cultured PBMCs also migrated in significantly higher numbers intocollagen gel containing IL-18 (500 ng/ml) than into gel containing mediumalone. Data are the mean � SEM of three experiments. �, p � 0.05; ��, p �0.01. More than 200 cells were scored in each experiment.

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observed in the absence of an IL-18 gradient (chemokinetic con-dition) or in the absence of IL-18 (spontaneous migration). Theseresults therefore demonstrate that IL-18 induced primarily a che-motactic response. We also compared the relative responsivenessof Th1 and Th2 cells to IL-18-induced polarization/cell migration.CD4� T cells from human cord blood were driven to Th1 or Th2lineages as described in Materials and Methods. Although rela-tively pure Th1 lines were easily obtained, a pure Th2 line proveddifficult to establish. Viability was low after two rounds of culti-vation. The Th2 lines retained a substantial and variable percent-age of IL-18R� IFN-� producing cells following a single round ofdriving under the Th2 condition. These cells polarized and mi-grated in response to IL-18. However, there was a marked decreasein the percentage of IL-18R� cells after the second round of driv-ing and this decrease was clearly reflected in the substantial de-crease in the percentage of cells migrating in response to IL-18(data not shown). It should be noted that although committed Th2cells are generally IL-18R, Th0 cells and differentiating Th2 cellsexpress a low level of IL-18R (29). Therefore, as much as IL-18R

is preferentially expressed on committed Th1 but not Th2 cells,IL-18 polarizes Th1 rather than Th2 cells. It should also be notedthat we have reported earlier that IL-18 attracts the migration ofneutrophils, which also express IL-18R (7).

Synovial lymphocytes activated in vivo exhibit chemotacticresponses to IL-18

SEB represents only a surrogate for physiologic activation of lym-phocytes. We therefore performed locomotion assays on lympho-cytes that were activated in vivo. IL-18 expression is up-regulatedin RA synovial tissues (5), particularly in lymphocyte-rich aggre-gates and adjacent to endothelia (Fig. 3). Mononuclear cells wereprepared from synovial fluids freshly aspirated from RA patientsand tested for their ability to polarize and migrate in the presenceof graded concentrations of IL-18. Lymphocytes polarized in re-sponse to IL-18 in a dose-dependent manner (Fig. 4a). Similarresults were obtained when lymphocytes were tested for their abil-ity to migrate through collagen gels (Fig. 4b). Synovial lympho-cyte fractions differ functionally and with respect to IL-18R� ex-pression. The expression of IL-18R� on CD4� and CD8�

synovial T cells was measured by FACS and revealed that whereas�50% of CD4� synovial T cells expressed IL-18R�, �10% ofCD8� T cells were IL-18R�� (Fig. 4, c and d). To investigatewhich cell subset(s) was the targets of the IL-18-mediated chemo-taxis ex vivo, immunofluorescence microscopy was used to iden-tify the responder cells. CD4�, but not CD8�, synovial T cellspolarize significantly to IL-18 compared with control medium(Fig. 4e). Moreover, CD19� synovial B cells did not express IL-

FIGURE 2. IL-18 induces migration of Th1 cells. Th1 cells were dif-ferentiated from CD4� T cells of human cord blood as described in Ma-terials and Methods. More than 90% of the cells expressed IL-18R� andproduced IFN-� but not IL-5. a, IL-18 induced Th1 cells migration intocollagen gel in a dose-dependent manner. Data are the mean � SEM ofthree experiments. �, p � 0.05; ��, p � 0.01 compared with IL-18-free gel.b, Time course of IL-18-induced migration. Th1 cells were overlaid on gelsincorporated with IL-18 (200 ng/ml) or medium alone. Gels were fixed atthe time points indicated and percentage of cells in the gel scored. Data arerepresentative of three experiments. c, The distance covered by migratingTh1 cells in IL-18 (200 ng/ml)-containing gel over time was determined bythe length of path (displacement from the starting position to the end point)covered by the cells. Data are the mean � SEM (n � 5). d, Chemotacticvs chemkinesis assays. Th1 cells were overlaid on gel that contained IL-18(200 ng/ml) or medium alone. The upper layer similarly contained IL-18(200 ng/ml) or medium alone. After 20 h, the percentage of cells in the gelwas scored and the results are presented as the mean � SEM of threeexperiments. ��, p � 0.01 compared with cultures containing medium/medium alone (first column).

FIGURE 3. Cells staining for IL-18 were observed within lymphoidaggregates and adjacent to blood vessel endothelium. a, IL-18-positivecells were detected by standard Immunohistology (brown) in formalin-fixed synovial membrane samples using anti-IL-18 Ab. This staining pat-tern could be neutralized by prior incubation of Ab with rIL-18, but notwith the related cytokine IL-1� (data not shown). b, No staining was ob-served with an irrelevant primary Ab acting as specific control. Originalmagnification, �40. Data are representative of 12 samples.

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18R� and did not respond to IL-18 in a polarization assay (data notshown).

IL-18 induces inflammatory cell recruitment in vivo

The above data suggested that IL-18 was a potent chemoattractantfor CD4� T cells in vitro, raising the possibility that IL-18 mayrecruit T cells into local tissues during inflammatory responses. Toinvestigate this, female DBA/1 mice were primed with P. acnesand then injected s.c. into a hind footpad with 500 ng of recom-

binant murine IL-18 or PBS alone. Mice injected with IL-18 de-veloped significant footpad swelling compared with PBS-injectedcontrols (Fig. 5). Moreover, IL-18 injection into footpads of IL-18-deficient mice, which express higher levels of IL-18R a priori(6, 10), led to significantly greater footpad swelling than IL-18-injected wild-type mice. Subsequent histologic analysis showeda substantial mononuclear infiltrate at the site of injection,whereas PBS-injected footpads were similar to noninjected con-trols (Fig. 6).

DiscussionIL-18 has recently been attributed numerous properties commen-surate with a vital role in innate and acquired immune responses.It is now clearly established that IL-18 can promote and regulatethe evolution of functionally distinct T cell subsets, driving towardeither type 1 or type 2 functional status dependent on ambientcytokine expression and costimulator expression on APC (3, 4).We now add to this the capacity to contribute to T cell recruitmentto inflammatory sites via promotion of chemotactic responses. Thismay be manifest particularly upon Th1 cells that retain IL-18R�expression as a phenotypic feature, although we cannot rule outsimilar effects manifest upon differentiating Th2 cells that tran-siently retain IL-18R� expression through earlier stages of matu-ration. “Resting” IL-18 expression at the mRNA and protein (atleast as pro-IL-18) levels has been observed in several tissues andcell types. By virtue of its early presence following tissue insult,the chemotactic properties demonstrated herein support a dual rolefor IL-18 in T cell activation. It may function as an importantmediator in determining the nature of memory T cells recruited(preferentially IL-18R��CD4� Th1 cells) at the same time as in-fluencing, in synergy with IL-12 or IL-4, the functional destinationof naive T cells present within a lesion. Importantly, we found noevidence of IL-18R� expression on naive human T cells derivedfrom cord blood nor any motile response by such naive cells toIL-18, and it is therefore unlikely that IL-18 has any significantrole in recruitment of such T cells.

FIGURE 5. IL-18 induces mononuclear cell migration in vivo. Groupsof intact (a, wild-type) mice and IL-18 KO DBA/1 (b) mice were injectedi.p. with P. acnes (500 �g/mouse) and injected 7 days later with recom-binant human IL-18 (500 ng/mouse) in the right footpad and PBS in the lefthind footpad. Footpad swelling was measured at regular intervals and ex-pressed as difference in the thickness between the right and left footpads.Data are the mean � SEM (n � 3) and are representative of three exper-iments. �, p � 0.05; ��, p � 0.01 compared with the corresponding PBScontrol group at each time point.

FIGURE 4. Synovial lymphocytes exhibit chemotactic response toIL-18 ex vivo. Mononuclear cells were prepared from synovial fluidsfreshly aspirated from RA patients and tested for their ability to polarizeand migrate in the presence of IL-18. a, Synovial mononuclear cells po-larized in response to IL-18 (30 min stimulation) in a dose-dependent man-ner. Data are the mean � SEM (n � 6). b, Freshly isolated synovial mono-nuclear cells were overlaid on collagen gel containing IL-18 (500 ng/ml)for 20 h and the percentage of invading cells was scored. Data are themean � SEM (n � 5). c, Flow cytometry analysis of the percentage ofCD4� and CD8� cells in the synovial mononuclear cell population andtheir expression of IL-18R� from a single patient. d, Mean � SEM of flowcytometry data from six patients. e, The percentage of polarized CD4� andCD8� synovial mononuclear cells in response to IL-18 (500 ng/ml, 30min) was determined by fluorescent microscopy. Cells (at least 200) werescored for each sample, and data are the mean � SEM (n � 4). �, p � 0.05;��, p � 0.01.

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It will be important to determine the relationship that IL-18-induced T cell recruitment has to that mediated by other estab-lished chemokine receptor-mediated pathways, in particular thoseoperating via CCR5, a putative Th1 cell marker (31). That IL-18-deficient mice generate inflammatory responses has been clearlydemonstrated in several models (6, 10), indicating that IL-18 ex-pression is not obligatory for host defense. Nevertheless, severalreports suggest that IL-18 deficiency does prejudice host responsesto some microbial species. Moreover, excess expression of IL-18is sufficient to initiate or considerably enhance autoimmune re-sponses. Transgenic cutaneous overexpression of caspase 1 in-duces high levels of IL-18 expression in the skin and leads todevelopment of a marked local inflammatory infiltrate that is de-pendent in large part upon the presence of IL-18 (32). Similarly,IL-18 injection into male MRL/lpr mice considerably acceleratesthe development of autoimmune glomerulonephritis and intrigu-ingly induces a pronounced cutaneous inflammatory infiltrate sim-ilar to that observed in caspase 1-transgenic mice (33). IL-18 ad-

ministration is sufficient to induce inflammatory arthritis incollagen/IFA-primed DBA/1 mice (34). We now show that localintroduction of recombinant mature IL-18 in vivo into a footpad issufficient to induce a marked mononuclear cell infiltrate. The pre-cise mechanisms whereby IL-18 mediates such effects in vivo requirefurther analysis. Although the in vitro time course results (IL-18-in-duced T cell polarization within 5 min) presented herein render itunlikely, our data do not exclude the possibility that IL-18 could op-erate through inducing release of intermediate factors, including che-mokines. This possibility is currently being investigated.

IL-18 may exert chemotactic responses in other cell lineages.Chemokinetic responses to IL-18 for endothelial cells may in partunderpin the proangiogenic effects of IL-18 in several neovascu-larization models (21). We have also shown that IL-18 may pro-mote neutrophil expression of adhesion molecule expression andrecruitment to the peritoneal cavity in vivo (7). IL-18 has also beenshown to up-regulate ICAM-1 expression on monocytes and on Tcells, providing further mechanisms whereby IL-18 can promote Tcell recruitment (35, 36).

Our data are of particular significance in determining the poten-tial activities of IL-18 in chronic inflammatory diseases such asRA. IL-18 detection in inflammatory sites is characterized by highlevels of expression in mononuclear cells adjacent to endothelialcells, providing circumstantial evidence that IL-18 is released in ageographically cogent area of an inflammatory lesion to facilitatefurther T cell recruitment (5). However, IL-18 could also operatewithin an inflamed tissue irrespective of trans-endothelial recruit-ment. We, and others, have recently proposed that recruitment ofmemory T cells and their subsequent cytokine-mediated activationwithin inflamed synovial membrane can drive downstream TNF-�release and consequent pathology (5, 37). Critical to this are cellcontact-dependent interactions between activated T cells and mac-rophages that in turn promote proinflammatory cytokine release(38). In unpublished studies we have shown that IL-18 can con-siderably enhance T cell/macrophage cell contact-mediated cyto-kine release (J. A. Gracie and I. B. McInnes, unpublished data). Itis attractive to propose that IL-18 released by macrophages gen-erates microgradients within synovial tissue that then optimizesfurther T cell recruitment and activation adjacent to the macro-phage, thereby perpetuating the chronic inflammatory process.

In conclusion, we provide here evidence that IL-18 has chemo-tactic activities in vitro and in vivo operating in particular uponhuman Th1 cells. Thus, in addition to its activities in functionalregulation of naive T cell differentiation, IL-18 may have an im-portant role in promoting recruitment of memory Th1 cells to aninflammatory lesion. This adds further to the portfolio of activitiesthat render IL-18 an attractive therapeutic target in a variety ofimportant autoimmune inflammatory disease states.

AcknowledgmentsWe thank Roderick Ferrier (Department of Pathology, Western Infirmary,Glasgow, U.K.) for technical assistance with histopathology. We alsothank Dr. J. Hunter (Gartnavel General Hospital, Glasgow, U.K.) for pro-viding additional synovial samples and Dr. Ian Mackay (Division of Im-munology, Infection and Inflammation, University of Glasgow, Glasgow,U.K.) for assistance in statistical analyses.

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FIGURE 6. Histology of the footpad of DBA/1 mice injected with PBS(a) or IL-18 (b) as in Fig. 5. The footpads were removed at 15 h afterinjection, fixed, and decalcified and 6-�m sections were stained with H&E.Footpads injected with IL-18 exhibited significant inflammatory infiltratecompared with the PBS control. Data are representative of six mice pergroup. Original magnification, �40. Inset in b shows lymphoid cells in theinfiltrate. Original magnification, �1000.

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