of April 9, 2019.This information is current as

γProtein-10 and Monokine Induced by IFN--InducibleγEosinophils: Role of IFN-

CXCR3 Expression and Activation of

PoulsenDissing, Hans-Jørgen Malling, Per S. Skov and Lars K.Reimert, Anders Millner, Sha Quan, Jens B. Hansen, Steen Tan Jinquan, Chen Jing, Henrik H. Jacobi, Claus M.

http://www.jimmunol.org/content/165/3/1548doi: 10.4049/jimmunol.165.3.1548

2000; 165:1548-1556; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/165/3/1548.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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CXCR3 Expression and Activation of Eosinophils: Role ofIFN-g-Inducible Protein-10 and Monokine Induced by IFN-g1

Tan Jinquan,2*‡ Chen Jing,*‡ Henrik H. Jacobi,* Claus M. Reimert,* Anders Millner,*Sha Quan,* Jens B. Hansen,* Steen Dissing,† Hans-Jørgen Malling,* Per S. Skov,* andLars K. Poulsen2*

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for bothIFN-g-inducible protein-10 (g IP-10) and monokine induced by IFN-g (Mig). We report a novel finding that CXCR3 is alsoexpressed on eosinophils.g IP-10 and Mig induce eosinophil chemotaxis via CXCR3, as documented by the fact that anti-CXCR3mAb blocks g IP-10- and Mig-induced eosinophil chemotaxis.g IP-10- and Mig-induced eosinophil chemotaxis are up- anddown-regulated by IL-2 and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA expressions in eosinophils are up-and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-timequantitative RT-PCR technique. g IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinaseA signaling pathways. The fact thatg IP-10 and Mig induce an increase in intracellular calcium in eosinophils confirms thatCXCR3 exists on eosinophils. Besides induction to chemotaxis,g IP-10 and Mig also activate eosinophils to eosinophil cationicprotein release. These results indicate that CXCR3-g IP-10 and -Mig receptor-ligand pairs as well as the effects of IL-2 and IL-10on them may be especially important in the cytokine/chemokine environment for the pathophysiologic events of allergic inflam-mation, including initiation, progression, and termination in the processes. The Journal of Immunology,2000, 165: 1548–1556.

Chemokines and their receptors are important elements forthe selective attraction and activation of various subsets ofleukocytes. The CXC chemokine receptor 3 (CXCR3),3 a G

protein-coupled, seven-transmembrane receptor, has been shown tobind IFN-g-inducible protein-10 (g IP-10) and monokine induced byIFN-g (Mig) with Ki values of 0.14 and 4.9 nM, respectively.g IP-10and Mig are two members of the CXC chemokine superfamily whoseexpression is dramatically up-regulated by IFN-g. Both chemokineshave been shown to be functional agonists of CXCR3 (1). The pro-teins act largely on NK cells and activated T cells and have beenimplicated in mediating some of the effects of IFN-g and LPS as wellas T cell-dependent anti-tumor responses. Recently, the CC chemo-kine 6Ckine and IFN-inducible T cella chemoattractant have been

identified as new ligands for CXCR3 (2, 3).g IP-10 and Mig inducerapid and transient adhesion of human IL-2-stimulated T lympho-cytes to immobilized integrin ligands through their receptorCXCR3, which is selectively expressed on activated T cells (4).Naive T cells expressed only CXCR4, whereas the majority ofmemory/activated T cells expressed CXCR3, and a small propor-tion expressed CCR3 and CCR5 (5). CXCR3 was expressed athigh levels on Th0 and Th1 lymphocytes and at low levels on Th2lymphocytes. In contrast, CCR3 and CCR4 were found on Th2lymphocytes (5). Circulating blood T cells, B cells, and NK cellsalso express CXCR3 (6). Blood T cells expressing CXCR3 weremostly CD45RO1 and generally expressed high levels ofb1 inte-grins. CXCR3 and CCR5 are markers for T cells associated withcertain inflammatory reactions, particularly Th1-type reactionssuch as rheumatoid arthritis. CXCR3 and CCR5 appear to identifysubsets of T cells in blood with a predilection for homing to thesesites (6). Interestingly, Mig was reported to promote tumor necro-sis in vivo (7).

In the present study we have observed thatg IP-10 and Miginduce eosinophil chemotaxis via CXCR3, as documented by thefact that anti-CXCR3 mAb blocksg IP-10- and Mig-induced eo-sinophil chemotaxis.g IP-10- and Mig-induced eosinophil chemo-taxis are up- and down-regulated by IL-2 and IL-10, respectively.Interestingly, CXCR3 protein and mRNA expressions in eosino-phils are up- and down-regulated by IL-2 and IL-10, indicating thatIL-2 and IL-10 controlg IP-10- and Mig-induced eosinophil che-motaxis via regulation of CXCR3 expression. It has been demon-strated thatg IP-10 and Mig induce eosinophil chemotaxis via thecAMP-dependent protein kinase A signaling pathway. The factthatg IP-10 and Mig induce an increase in [Ca21]i in eosinophilsconfirms that CXCR3 exists on eosinophils. Besides induction tochemotaxis,g IP-10 and Mig also activate eosinophils to eosino-phil cationic protein (ECP) release. These results indicate thatCXCR3-g IP-10 and -Mig receptor-ligand pairs and modulation ofCXCR3 expression by IL-2 and IL-10 may be especially important

*Laboratory of Medical Allergology, Allergy Unit, National University Hospital, and†Department of Medical Physiology, University of Copenhagen, Copenhagen, Den-mark; and‡Department of Immunology, Anhui Medical University, Hefei, People’sRepublic of China

Received for publication October 5, 1999. Accepted for publication May 17, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Danish Allergy Research Center(to T.J., A.M., and S.Q.), a grant from H:S Direktionen (Copenhagen, Denmark; toC.J.), the Alfred Benzons Foundation (to T.J.), and a grant from Novo Nordisk A/S(Copenhagen, Denmark; to S.Q.) and in part by the Simon Fougner Hartmanns Foun-dation and the National Science Foundation of China (no. 39870674).2 Address correspondence and reprint requests to Dr. Tan Jinquan or Dr. Lars K.Poulsen, Laboratory of Medical Allergology, National University Hospital, DK-2200Copenhagen N., Denmark. E-mail addresses: tan@rh.dk or lkpallgy@inet.uni2.dk3 Abbreviations used in this paper: threshold cycle; CXCR, CXC chemokine receptor;CT, threshold cycle; BIM I, bisindolylmaleimide I; C. I., chemotactic index; ECP,eosinophil cationic protein; [Ca21]i, intracellular calcium; H-8,N-(2-(methylamin-o)ethyl)-5-isoquenolinesulfonamide dihydrochloride; H-89,N-(2-(r-bromocin-namylamino)ethyl)-5-isoquenilesulfonamide;g IP-10, IFN-g-inducible protein-10;a-IL-2R, anti-IL-2R; MCNC, migrating cells on negative control; Mig, monokineinduced by IFN-g; MPC, magnetic particle concentrator; PKA, protein kinase A;PKC, protein kinase C; PTK, protein tyrosine kinase; PT, pertussis toxin; Sta,staurosporine.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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in the cytokine/chemokine environment that controls initiation,progression, and termination of allergic and other eosinophil-dom-inated forms of inflammation processes.

Materials and MethodsPurification and treatment of eosinophils

Human peripheral eosinophils were purified from healthy, nonallergic vol-unteers as described in detail previously (8, 9). Briefly, the method wasbased on Percoll gradient centrifugation (density, 1.082 g/ml; Pharmacia,Uppsala, Sweden) to isolate granulocytes, lysis of RBC with 155 mMammonium chloride (NH4Cl), and immunomagnetic depletion of neutro-phils by the magnetic cell separation system (MACS) using anti-CD16-coated MACS particles (Miltenyi Biotech, Bergisch Gladbach, Germany).The purity of eosinophil preparations was invariably$97%, as judged byeosin staining. Throughout the purification procedure, the cells were keptat 4°C in a Ca21- and Mg21-free medium.

Stimulation of cells

For chemotaxis regulation, purified eosinophils were preincubated withTh1-associated cytokines (IL-2, IFN-g, or TNF-a; optimal concentration at10 ng/ml) (10, 11) and Th2-associated cytokines (IL-4, IL-5, or IL-10;optimal concentration at 10 ng/ml) (11) for 24 h at 37°C in 5% CO2 beforechemotaxis assay. For measurement of CXCR3 mRNA, purified eosino-phils were preincubated with IL-2 (10 ng/ml) or IL-10 (10 ng/ml) for 24 hat 37°C. Then the cells were subjected to mRNA isolation for real-timequantitative RT-PCR. All cytokines used were purchased from R&D Sys-tems Europe (Abingdon, U.K.). For investigation of signaling pathway ofg IP-10 and Mig to induce eosinophil chemotaxis, the cells were preincu-bated for 45 min at room temperature with pertussis toxin (PT; 1mg/ml),staurosporine (Sta; 1mM), tyrphostin 23 (1 mM), N-(2-(methylamin-o)ethyl)-5-isoquenolinesulfonamide dihydrochloride (H-8; 30mM), N-(2-(r-bromocinnamylamino)ethyl)-5-isoquenilesulfonamide (H-89; 30mM),or bisindolylmaleimide I (BIM I; 1mM), respectively, before the cells weresubjected to additional experiments. All signaling pathway inhibitors usedwere purchased from Sigma (St. Louis, MO). For measurement of ECP, thepurified human eosinophils were stimulated with chemokines at differentconcentrations, as indicated, for 4 h at 37°C in 96-well plates. The super-natants were collected after stimulation for ECP measurement.

Chemotaxis assay

The following human recombinant chemokines were studied:g IP-10, Mig,and eotaxin (R&D Systems Europe). The chemotaxis assay was performedusing a 48-well microchamber (Neuro Probe, Bethesda, MD) technique(12). Briefly, chemokines were diluted in RPMI 1640 with 0.5% pooledhuman serum and placed in the lower wells (25ml). Fifty microliters of thecell suspension at 13 106 cells/ml was added to the upper well of thechamber, which was separated from the lower well by a 5-mm pore size,polycarbonate, polyvinylpyrolidone-free membrane (Nucleopore, Pleasan-ton, CA). The cells were freshly isolated eosinophils or eosinophils incu-bated with reagents as indicated. The chamber was incubated for 60 min at37°C in an atmosphere containing 5% CO2. The membrane was then care-fully removed, fixed in 70% methanol, and stained for 5 min in 1% Coo-massie brilliant blue. The cells that migrated and adhered to the lowersurface of the membrane were counted using a light microscopy. Approx-imately 6% of eosinophils will migrate spontaneously (known as migratingcells on negative control, MCNC) (10), corresponding to between 2500 and4000 cells. It may vary from day to day, but very little within the sameday’s experiments. The results were expressed as a chemotactic index(C. I.), which is the ratio between the number of migrating cells in thesample and that in the medium control (12), and with the SD. For blockingtests of unstimulated or stimulated eosinophil chemotaxis toward the che-mokines indicated, the cells were preincubated with either anti-CXCR3mAb (5 mg/ml; clone 49801.111, R&D Systems, Oxon, U.K.) or isotypeIgG1 (5 mg/ml) for 60 min at room temperature before chemotaxis assay.

Flow cytometry

As previously described (13), eosinophils either freshly isolated or stimu-lated with cytokines were first incubated with a mouse anti-human CXCR3mAb or anti-CCR3 mAb (R&D Systems Europe, clone 49801.111; or Leu-kocite, Cambridge, CA, clone 7B11) at 5mg/ml or with 5mg/ml matchedisotype mouse IgG1 (Dako, Glostrup, Denmark) in PBS containing 2%BSA and 0.1% sodium azide for 20 min. The cells were then washed twicein staining buffer and resuspended in FITC-conjugated F(ab)2 donkey anti-mouse mAb (1/250, v/v; Jackson ImmunoResearch Laboratories, WestGrove, PA) for 20 min, followed by washing twice in staining buffer. All

procedures were conducted at 4°C. The cells were then fixed with 1%paraformaldehyde. The analyses were performed with a flow cytometer(Coulter XL; Coulter, Miami, FL).

Immunocytochemistry assay

For detection of chemokine receptors on eosinophils, the freshly isolatedresting cells were spun down on a glass slide at 800 rpm for 4 min. Thenthe slide was moved into a fixation dish immediately to avoid drying out.The fixation liquid was a mixture of methanol and acetone (1/1, v/v). After5-min fixation the preparation was washed twice in PBS for 5 min. Block-ing buffer (PBS with 1% BSA and 0.3% Triton X-100) was added for 5 minat 20°C to avoid unspecific binding, followed by primary Ab (a mouseanti-human CXCR3 mAb (R&D Systems Europe, clone 49801.111) orCCR3 mAb (Leukocite, clone 7B11) at concentration of 10mg/ml. Thepreparation was incubated overnight at 4°C. The next day the preparationwas washed twice in PBS for 5 min each time, followed by addition ofsecondary Ab and was visualized using the alkaline phosphatase stainingsystem (Dako) according to the manufacturer’s instruction. Finally, thepreparation was sealed and stored in the dark until observation under amicroscope.

Real-time quantitative RT-PCR assay

All real-time quantitative RT-PCR reactions were performed as describedpreviously (9, 14, 15). Briefly, total RNA from peripheral eosinophils (13106; purity, .99%) was prepared using the Quick Prep Total RNA Ex-traction Kit (Pharmacia Biotech, Piscataway, NJ), and any potential con-taminating chromosomal DNA was digested with DNase I according to themanufacturer’s instructions. For RT, the RNA was reverse transcribed us-ing oligo(dT)12–18 and Superscript II reverse transcriptase (Life Technol-ogies, Grand Island, NY), according to the manufacturer’s instructions. RTwas performed for 60 min at 37°C, and any potential contaminating proteinwas denatured by incubation for 10 min at 95°C. The real-time quantitativePCR was performed in special optical tubes in a 96-well microtiter plate(Perkin-Elmer/Applied Biosystems, Foster City, CA) with an ABI PRISM7700 Sequence Detector Systems (Perkin-Elmer/Applied Biosystems), ac-cording to the manufacturer’s instructions. By using the SYBR Green PCRCore Reagents Kit (Perkin-Elmer/Applied Biosystems, P/N 4304886), flu-orescence signals were generated during each PCR cycle via the 59- to39-endonuclease activity of AmpliTaq Gold (14) to provide real-time quan-titative PCR information. The CXCR3 genes were generated by connectingthe following sequences of the specific primers (purchased from DNATechnology, Aarhus, Denmark): sense, 59-GGAGCTGCTCAGAGTAAATCAC-39; and antisense, 59-GCACGAGTCACTCTCGTTTTC-39.

All unknown cDNAs were diluted to contain equal amounts ofb-actincDNA. The standards, no template controls, and unknown samples wereadded in a total volume of 50ml/reaction. PCR retain conditions were 2min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 60 s at 60°Cfor each amplification. Potential PCR product contamination was digestedby uracil-N-glycosylase, because dTTP is substituted by dUTP (14). AllPCR experiments were performed with a hot start. In the reaction system,uracil-N-glycosylase and AmpliTaq Gold (Perkin-Elmer/Applied Biosys-tems) were applied according to the manufacturer’s instructions (14, 15).To analyze data for PCR products two terms were used to express theresults:DRn, the normalized reporter signal minus the baseline signal es-tablished in the first few cycles PCR; and CT (threshold cycle), the PCRcycle at which an increase in reporter fluorescence signal above a baselinecan first be detected.

Changes in [Ca21] i in single eosinophils

[Ca21]i in single eosinophils was measured as described previously (16).Briefly, purified human eosinophils (13 105 cells/ml) were loaded withfura-2/AM at 2 mM at 37 C° for 30 min in PIPES buffer. One-millilitereosinophil suspensions were placed in a specially constructed chamber(Nunc, Roskilde, Denmark) without any coating for cell attachment. Thechanges in [Ca21]i were determined using a digital imaging system con-sisting of a Zeiss Axiovert 135 microscope (New York, NY), a light-sen-sitive video camera (Genesys, DAGE MTI, Michigan City, IN), and soft-ware from Universal Imaging (Media, PA). The stimuli were added at theconcentrations indicated. The [Ca21]i concentrations were recorded in theindividual cells before and after stimulation. Ionomycin stimulation (1 nM)was followed to record the maximal increase in [Ca21]i. The gray scalevalues from each cell were converted into [Ca21]i by dividing fluorescenceemissions measured following 340 and 380 nm excitation. TheRmax valuewas 1.1, and theRmin was 0.1. TheKd for fura-2 was 224 nM, and theproportionality coefficient, sf2/sb2, measured as the fluorescence intensityexciting at 380 nm from the solutions containing low concentrations of freeand Ca21-saturated dye amounting to 2.0, was 2.

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ECP release assay

The eosinophil cationic protein release assay was performed as describedpreviously (17). Briefly, about 10,000 eosinophils were incubated in heat-stable Ag-coated microtiter plates for 4 h at 37°C, followed by harvestingof supernatants. ECP in supernatants and cell extracts were quantified by asolid phase sandwich ELISA method with a biotin-avidin amplificationsystem in microtiter plates (Nunc). Each well in the microtiter plates wascoated with 100ml of rabbit anti-ECP polyclonal Ab (1.5mg/ml) overnightat 4°C. Before use, the plates were washed three times. The ECP standardswere calibrated using an extinction coefficient, E1%

1cm, of purified ECP at280 nm5 15.45, i.e., a 1% solution of the protein has absorbance of 15.45when using a light path of 1 cm. The samples were incubated overnight,followed by addition of biotin-conjugated rabbit anti-ECP polyclonal Abfor 1.5 h at 37°C, and then were exposed to avidin-peroxidase at roomtemperature for 30 min, followed by enzyme reaction for 20 min. Theabsorbance was measured at 492 nm with a 620 nm reference. The per-centage of release was the ratio between free ECP in the supernatant andthat in the total cell extract.

Resultsg IP-10 and Mig induce eosinophil chemotaxis via CXCR3

We examined the abilities ofg IP-10 and Mig to induce eosinophilchemotaxis.g IP-10 and Mig have induced significant eosinophilchemotactic migration. The results in Fig. 1A show thatg IP-10and Mig induced chemotactic migration in freshly isolated eosin-ophils, yielding typical bell-shaped dose-dependent chemotaxis re-sponse curves. The optimal chemotactic concentrations ofg IP-10and Mig were both 100 ng/ml (C. I., 61 4.16 0.37 and 4.26 0.52,

respectively; MCNC, 31046 766). Eotaxin was used as positivecontrol and induced a similar eosinophil chemotactic migration(C. I., 61 2.46 0.26). To confirm that the observed eosinophilchemotaxis was indeed induced byg IP-10 and Mig via CXCR3,we used anti-CXCR3 mAb to block the eosinophil chemotacticactivity of g IP-10 and Mig. The anti-CXCR3 mAb could com-pletely block the chemotaxis of eosinophils towardg IP-10 andMig (Fig. 1B; C. I., 61 1.16 0.12 and 0.96 0.13, respectively;MCNC, 37456 359; both at 100 ng/ml), whereas it did not in-terfere the chemotaxis of eosinophils toward eotaxin (C. I., 613.4 6 0.42). The anti-CCR3 mAb completely blocked eosinophilchemotaxis toward eotaxin (data not shown). The isotype Ab hadno blocking effect (Fig. 1C; C. I., 61 3.16 0.38, 3.86 0.53, and3.9 6 0.47, respectively; MCNC, 3, 8246 479), The results ofcheckerboard analysis (18) demonstrate that migratory movementsof eosinophils towardg IP-10 and Mig are chemotactic, but notchemokinetic (data not shown).

g IP-10- and Mig-induced eosinophil chemotaxis are regulatedby IL-2 and IL-10

We also examined the abilities of Th1- and Th2-associated cyto-kines to regulateg IP-10- and Mig-induced eosinophil chemotaxis.The results in Table I show that Th1-associated cytokine IL-2 sig-nificantly up-regulatedg IP-10- and Mig-induced eosinophil che-motaxis (C. I., 61 6.206 0.45 and 6.226 0.39, respectively;

FIGURE 1. The migration of freshly isolated (A), anti-CXCR3 mAb-incubated (B), or isotype mouse IgG1-incubated (C) eosinophils towardg IP-10(f), Mig (M), or eotaxin (v). The illustrated data are from a single representative experiment of four performed. All results were determined as describedin Materials and Methodsand are expressed as the chemotactic index (C.I.), based on triplicate determinations of chemotaxis with each concentration ofchemoattractant. The applied chemokine concentrations (C.C.) are indicated as nanograms per milliliter.p, Statistically significant difference (allp , 0.001)in freshly isolated eosinophil chemotaxis towardg IP-10 and Mig vs anti-CXCR3 mAb-pretreated eosinophil chemotaxis towardg IP-10 and Mig(A vs B) as well as in chemotaxis of eosinophils toward eotaxin vs chemotaxis of eosinophils towardg IP-10 and Mig (B). Otherwise, there is nostatistical significant difference (allp . 0.05) at corresponding chemokine concentrations.

Table I. Effect of Th1- and Th2-associated cytokines on chemokine-induced eosinophil chemotaxisa

Chemokineb None IL-2c IFN-g TNF-a IL-4 IL-5 IL-10

Expt. 1g IP-10 2.626 0.34d 6.206 0.45* 2.376 0.33 2.476 0.24 2.516 0.24 2.216 0.24 1.146 0.09*Mig 2.386 0.27 6.226 0.39* 2.256 0.41 2.506 0.23 2.416 0.55 2.596 0.75 1.186 0.15*Eotaxin 2.716 0.55 2.456 0.28 2.606 0.29 2.286 0.51 2.396 0.41 2.526 0.30 2.416 0.27MCNC 36016 674 33976 547 27346 393 32386 626 37256 368 26826 899 28736 792

Expt. 2g IP-10 2.496 0.18 5.106 0.44* 2.396 0.58 2.506 0.57 2.386 0.53 2.656 0.49 1.396 0.18*Mig 2.466 0.27 5.726 0.51* 2.366 0.64 2.496 0.39 2.446 0.35 2.506 0.18 1.206 0.21*Eotaxin 2.516 0.51 2.436 0.36 2.456 0.67 2.326 0.25 2.546 0.69 2.896 0.23 3.286 0.62MCNC 40146 364 32486 543 38216 688 31876 391 29816 782 35656 487 29166 453

a The eosinophils were isolated from venous blood from healthy volunteers and incubated with Th1- and Th2-associated cytokines for 24 h before chemotaxis assay.b The chemokines applied were all at 100 ng/ml in optimal concentrations.c Th1- and Th2-associated cytokines were applied all at 10 ng/ml in optimal concentrations.d All listed data were determined as described inMaterials and Methodsand expressed as chemotactic index (C.I.)6 S.D., and based on triplicate determination of chemotaxis

on each concentration of chemoattractant.* , Statistical significant difference (allp , 0.001) in freshly isolated eosinophil chemotaxis vs Th1- or Th2-associated cytokine-treated eosinophil chemotaxis. Otherwise,

there is no statistical significant difference (allp . 0.05).

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MCNC, 3397 6 547), whereas other Th1-associated cytokinesIFN-g and TNF-a showed no such effect. Moreover, Th2-associ-ated cytokine IL-10 significantly down-regulated chemotactic mi-gration of eosinophils induced byg IP-10 and Mig (C. I., 611.146 0.09 and 1.186 0.15, respectively; MCNC, 28736 792),whereas other Th2-associated cytokines, IL-4 and IL-5, showed nosuch effect. None of the tested Th1- and Th2-associated cytokineshad modulated eosinophil chemotaxis toward eotaxin (100 ng/ml).The regulatory effects of IL-2 and IL-10 on chemotactic migrationof eosinophils induced byg IP-10 and Mig could be blocked byanti-IL-2R mAb (a-IL-2Ra) and anti-IL-10R mAb, respectively(data not shown).

Expression of CXCR3 on eosinophil is regulated by IL-2 andIL-10

The results from flow cytometric analyses in Fig. 2 document thatthere were about 48.3% CXCR31 cell fractions in freshly isolatedeosinophils (Fig. 2B). After 24-h incubation with cytokine-freemedium, there was no significant change in the CXCR31 cell frac-tion (41.6%; Fig. 2C). Interestingly, IL-2, a Th1-associated cyto-kine significantly up-regulated the expression of CXCR3 on eo-

sinophils by up to 98.8% (Fig. 2D). IL-10, a Th2-associatedcytokine, showed a robust ability to down-regulate the expressionof CXCR3 on human peripheral eosinophils. After 24-h incubationwith IL-10 (10 ng/ml), CXCR31 eosinophil were reduced to 5.4%(Fig. 2E). CCR3 were constantly expressed on eosinophils despitethe stimulation with IL-2 and IL-10. There were 99.6% CCR3-positive cells in freshly isolated eosinophils (Fig. 2G), 99.5% ineosinophils cultured in cytokine-free medium (Fig. 2H), and98.2% in IL-2-stimulated eosinophils (Fig. 2I) and IL-10-stimu-lated eosinophils (Fig. 2J). Fig. 2,A andF, shows isotype controlsfor CXCR3 and CCR3 mAbs, respectively. Interestingly, we foundthat the freshly isolated and stimulated eosinophils are either pos-itive or negative, with no gradations in the level of CXCR3 ex-pression. The explanation may be that the positive cells areabun-dantly expressed CXCR3, whereas the negative cells are absolutelynot expressed CXCR3, or that CXCR3 on these cells are absoluteat a detectable level on flow cytometer. IL-2 and IL-10 play rolesto switch on or switch off CXCR3 expression on the cells. Weconducted similar experiments on T lymphocytes, but did not findup- or down-regulation of CXCR3 expression on these cells (datanot shown). The regulatory effects of IL-2 and IL-10 on CXCR3expression on eosinophils can be blocked by anti-IL-2R mAb (a-IL-2 Ra) and anti-IL-10R mAb, respectively (data not shown).

FIGURE 2. Single-color flow cytometric analysis of the distributionand modulation of CXCR3 and CCR3 on eosinophils. For CXCR3 detec-tion, the cells were either freshly isolated (B) or stimulated for 24 h withcytokine-free medium (C), IL-2 (D), and IL-10 (E), respectively. ForCCR3 detection, the cells were either freshly isolated (G) or stimulated for24 h with cytokine-free medium (H), IL-2 (I), and IL-10 (J), respectively.A andF, Isotype IgG1 Ab controls for CXCR3 and CCR3, respectively.The percentages of CXCR31 and CCR31 cells are indicated inResults.The data are from a single experiment, which is representative of six sim-ilar experiments performed. In every measurement;73% of 10,000 ac-quired events were gated and estimated. There is a statistically significantdifference (allp , 0.0001) in CXCR31 cells forB vs D or E. There is nostatistical significant difference (allp . 0.05) in CXCR31 cells forB vs C.There is no statistical significant difference (allp . 0.05) in CCR31 cellsfor G vs H, I, or J.

FIGURE 3. Expression of CXCR3 (B) and CCR3 (C) on human pe-ripheral eosinophils. The cells were freshly isolated from a healthy donorwith purity invariably $97% as judged by eosin staining, fixed with amixture of methanol and acetone (1/1, v/v), and immunostained as de-scribed inMaterials and Methods. Immunoreactive cells were visualizedusing an alkaline phosphatase staining system.B, The cells were stainedwith primary Ab of anti-CXCR3;C, cells were stained with primary Ab ofanti-CCR3;A, cells were stained with a negative control isotype Ab. Cellswere photographed under3400 magnification. Bar5 ;10 mm.

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IL-2R was demonstrated to be expressed on human eosinophils(19). Although there is no direct evidence of the existence of IL-10R on human eosinophils, IL-10 was shown to directly functionon human eosinophils (20, 21). Our results demonstrate that twocytokines, via their receptors on eosinophils, act to regulate theexpression of CXCR3 on the cells and to further modulate thebiological function ofg IP-10 and Mig on the eosinophils. Toconfirm that expression of CXCR3 is indeed on the eosinophilsand to completely rule out the possibility that the observed effectsmay be due to contaminating lymphocytes, we therefore conductedimmunocytochemical assay on purified eosinophils to demonstratethe existence of CXCR3 and CCR3 (as a positive control) on eo-sinophils. Because there is autofluorescence in eosinophils, wehave chosen an alkaline phosphatase staining system for visual-ization in the immunocytochemical assay to be absolutely sure ofthe observed positive results. The results from the immunocyto-chemical assay document that CXCR3 is expressed in the humanperipheral eosinophils (Fig. 3B), as well as that CCR3 is expressed(Fig. 3C). Fig. 3Ashowd the mouse isotype Ab-negative control.We also conducted similar experiments on T lymphocytes, and wefound CXCR3 expression on these cells (22), but not CCR3 ex-pression (data not shown). It should be stressed that we used pu-rified eosinophils with a purity invariably$97% as judged byeosin staining. Morphologically, the CXCR3-positive cells wereidentified as eosinophils in the immunocytochemical assay.

CXCR3 mRNA expression in eosinophils is regulated by IL-2and IL-10

The results in Fig. 4Ashow that mRNA of CXCR3 was detectedin human peripheral resting eosinophils. Compared with the am-plification of standard DNA template (2.03 104 copies) with a

housekeeping gene (b-actin), there were;2.0 3 103 copies forCXCR3 in the tested samples of resting eosinophils. There were;1.5 3 104 copies for CXCR3 in the tested samples of IL-2-stimulated eosinophil in 24 h. There were;6.9 3 102 copies forCXCR3 in the tested samples of IL-10-stimulated eosinophils in24 h. The results in Fig. 4B show that a linear relationship betweenthe threshold cycle, CT, and the log starting quantity of standardDNA template or target cDNA (CXCR3) was detected. In all ex-periments the correlation coefficient was;0.93. Because eosino-phils can be very fragile cells, we tested the viability of eosinophilsafter stimulation with IL-2 or IL-10 (all at 10 ng/ml). The viabilityof the cells was invariably$95%, as determined by trypan blueexclusion test. This experiment was able to exclude the possibilitythat the cells studied after 24-h stimulation with IL-2 or IL-10 aremerely a subpopulation of the input cells.

FIGURE 4. The plots of the real-time detection and amplification ofmRNA of CXCR3 in unstimulated, IL-2-stimulated, and IL-10-stimulatedeosinophils.A, Black, amplification of mRNA of CXCR3 in unstimulatedeosinophils; blue, amplification of CXCR3 mRNA of IL-2-stimulated eo-sinophils; orange, amplification of CXCR3 mRNA of IL-10-stimulated eo-sinophils; and red, amplification of standard DNA template (2.03 104

copies) with a housekeeping gene (b-actin). CT values were 17.016 forstandard DNA template, 21.926 for mRNA of CXCR3 in unstimulatedeosinophils, 18.598 for mRNA of CXCR3 in IL-2-stimulated eosinophils,and 26.220 for mRNA of CXCR3 in IL-10-stimulated eosinophils.B, Thelinear relationship between CT and log starting quantity (S.Q.) of standardDNA template (black circles) or target (CXCR3) mRNA (red circles). Theplots shown are representative of two similar experiments conducted.

FIGURE 5. The changes in [Ca21]i in a single human eosinophil. Thefura-2-loaded eosinophils were stimulated withg IP-10 (A), Mig (B),eotaxin (C), org IP-10, and consequently Mig (D), as indicated. The stim-uli concentrations were all about 50 ng/ml as described inMaterials andMethods. Ionomycin was applied at final concentration of 0.1 nM. The[Ca21]i-dependent fluorescence changes are shown. All stimuli were testedat least twice with cells from different unselected donors.

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Involvement of signaling pathways ing IP-10- and Mig-inducedeosinophil chemotaxis

To explore which signaling pathways are involved ing IP-10- andMig-induced eosinophil chemotaxis, we examined whether the in-terference with different signaling pathways can affect eosinophilchemotaxis towardg IP-10 and Mig. Before the chemotaxis assay,we pretreated eosinophils for 30 min at 37°C with tyrphostin 23(1 mM), a selective inhibitor of PTK (23); Sta (1mM), a selectiveinhibitor of protein kinase (24); H-89 (30mM), a selective inhib-itor of cAMP-dependent protein kinase (25); PT (1mg/ml), a spe-cific inhibitor of certain G proteins (26); H-8 (30mM), a selectiveinhibitor of cAMP- and cGMP-dependent protein kinase A (27); orBIM I (1 mM), a selective inhibitor of protein kinase C (28), re-spectively. Table II shows thatg IP-10- and Mig-induced eosino-phil chemotaxis can completely and selectively be blocked bystaurosporine and H-89 at applied concentrations, whereaseotaxin-induced eosinophil chemotaxis (which is via its receptorCCR3) can only be blocked by Sta, not by H-89. Eotaxin-inducedeosinophil chemotaxis (which is via its receptor CCR3) can com-pletely and selectively be blocked by tyrphostin 23, but not by Staand H-89. The doses of PT or BIM I (see above andMaterials andMethods) employed did not changed the pattern ofg IP-10-, Mig-,and eotaxin-induced eosinophil chemotaxis. Sta and H-89 wereselective inhibitors of protein kinase or selective inhibitors ofcAMP-dependent protein kinase A, respectively, and tyrphostin 23was a selective inhibitor of PTK, respectively. Thus, these resultsstrongly indicate thatg IP-10 and Mig induce eosinophil chemo-taxis via the cAMP-dependent protein kinase A signaling path-ways, and that eotaxin (which is via its receptor CCR3) induceseosinophil chemotaxis via a PTK signaling pathway. We pro-longed the incubation time of the cells with PT up to 2 h. Thepretreated cells still showed the same ability to migrate towardg

IP-10, Mig, and eotaxin, which confirms that neitherg IP-10, Mignor eotaxin induces eosinophil chemotaxis via certain G proteinssignaling pathways (data not shown). In view of the limited spec-ificity of applied signaling pathway inhibitors, more complemen-tary experiments are needed to confirm the observed effects ofthese inhibitors. It must be mentioned that H-8, a selective inhib-itor of cAMP- and cGMP-dependent protein kinase A, has failed toinhibit g IP-10- and Mig-induced eosinophil chemotaxis at theconcentration used in our experiments. This controversy requiresfurther clarification.

g IP-10 and Mig induce an increase in [Ca21] i in eosinophils

There is an immediate increase in [Ca21]i after CXCR3 ligandsgIP-10 (final concentration, 50 ng/ml) and Mig (final concentration,50 ng/ml) stimulation (Fig. 5,A andB). Eotaxi, which is known toactivate eosinophils, has been used as a positive control (Fig. 5C).A maximal increase in [Ca21]i has been demonstrated by the ad-dition of ionomycin (0.1 nM) after stimulation withg IP-10 andeotaxin. Thus, the results indicate that the ligandsg IP-10 and Migbind to their common receptor CXCR3 on eosinophils to induce[Ca21]i changes in eosinophils. In cross-desensitization in the cal-cium flux betweeng IP-10 and Mig,g IP-10 can significantlydesensitize Mig in terms of calcium flux in eosinophils (Fig. 5D)and vice versa (data not shown). These data further support theidea thatg IP-10 and Mig act through the same receptor CXCR3.

g IP-10 and Mig induce ECP release

We also examined ECP release from human peripheral eosino-phils. The results in Fig. 6 document that about 2.0% ECP are

Table II. Blocking effect of signaling pathway inhibitors on chemokine-induced eosinophil chemotaxisa

Inhibitorb

gIP-10c Mig Eotaxin

MCNC100 101 102 103 100 101 102 103 100 101 102 103

None 1.23d 2.36 3.41 1.13 0.99 2.68 2.75 0.98 0.92 1.95 2.71 0.89 36236 791PT 1.21 2.85 3.51 1.39 1.11 3.31 3.13 0.83 1.24 2.96 3.31 1.09 29616 397Sta 1.21 1.59* 1.81* 1.39 1.39 1.26* 1.36* 1.32 1.36 1.91 2.55 1.32 31216 556Ty 23 1.14 2.55 3.05 1.54 1.26 2.35 2.63 1.19 1.26 1.31* 1.15* 1.22 37656 361H-8 1.31 2.98 3.15 1.31 1.21 2.36 3.16 0.97 1.23 3.13 2.89 1.11 25696 768H-89 1.37 1.21* 1.35* 1.07 1.02 0.94* 0.71* 1.31 1.15 2.83 3.11 1.22 40076 752BIM I 1.31 2.65 3.25 1.25 1.09 2.83 3.25 1.18 1.26 2.99 3.13 1.35 28726 653

a The eosinophils were isolated from venous blood from healthy volunteers and incubated with signaling pathway inhibitors for 45 min before chemotaxis assay.b The concentrations of signaling pathway inhibitors applied were indicated inMaterials and Methods.c The concentrations of chemokines were applied in ng/ml.d All listed data were determined as described inMaterials and Methodsand expressed as chemotactic index (C.I.)6 S.D., and based on triplicate determination of chemotaxis

on each concentration of chemoattractant. The listed data are from a single representative experiment of three performed.* , Statistical significant difference (allp , 0.001) in freshly isolated eosinophil chemotaxis vs different signaling pathway inhibitor-treated eosinophil chemotaxis (chemo-

kines at 10 ng/ml and 100 ng/ml). Otherwise, there is no statistical significant difference (allp . 0.05) at corresponding chemokine concentrations. For simplifying the table,the SDs are not indicated, but all SDs vary between 0.19 and 0.56.

FIGURE 6. The release of ECP after stimulation withg IP-10, Mig, oreotaxin at different concentrations (nanograms per milliliter) as indicated.The different symbols (f,M, andv) represent amounts of ECP release(nanograms per milliliter) from the different individuals tested. One hun-dred microliters of human eosinophil suspension (13 105 cells/ml) waseither unstimulated or stimulated with chemokines indicated for 4 h at37°C in 96-well plates as described inMaterials and Methods. The super-natants were collected after stimulation. ECP content was measured asdescribed inMaterials and Methods.p, Statistically significant difference(all p , 0.001) in amounts of spontaneously ECP release of eosinophils vsthat induced byg IP-10, Mig, or eotaxin at different concentrations. Oth-erwise, there is no statistically significant difference (allp . 0.05).

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spontaneously released during the 4-h culture with cytokine-freemedium (;760 ng/ml in total in each cell aliquot in our system).After 4-h incubation withg IP-10, there was significant ECP re-lease in a dose-dependent manner. At 13 103 ng/ml, it induced41.7% ECP release. After 4-h incubation with Mig, it inducedsignificant ECP release in dose-dependent manner. At 13 103

ng/ml, it induced 38.1% ECP release. After 4-h incubation witheotaxin, there was significant ECP release in dose-dependent man-ner. At 13 103 ng/ml, it induced 39.1% ECP release. There wereno significant differences among the three chemokines.

DiscussionChemokines and their receptors are important in cell migrationduring inflammation, in the establishment of functional lymphoidmicroenvironments, and in organogenesis. Chemokines direct tis-sue invasion by specific leukocyte populations. CXCR3 is notbroadly expressed in cells of the immune system, except about40% of resting T lymphocytes and low numbers of B cells and NKcells were stained positively for CXCR3 (29). Interestingly, thesecells did not express CXCR3 transcripts and did not respond tothese chemokines. Pretreatment with IL-2 resulted in cultures offully responsive, CXCR3-positive T lymphocytes (30). To date,CXCR3 has been identified as the receptor forg IP-10, Mig,6Ckine, and IFN-inducible T cella chemoattractant (2, 3, 30). Dueto the importance of CXCR3 and its ligands in immunity, a numberof interesting and intensive studies have appeared recently. It hasbeen suggested that CXCR3 and CCR5 are preferentially ex-pressed in human Th1 lymphocytes, whereas Th2 lymphocytespreferentially express CCR4 and CCR3 (31). CXCR3 has beendocumented to be expressed on lymphocytic cells in virtually ev-ery perivascular inflammatory infiltrate in active multiple sclerosislesions, and cerebrospinal fluid T cells are significantly enrichedfor cells expressing CXCR3 or CCR5 (32).g IP-10 even plays arole in the epidermotropism of cutaneous T cell lymphoma (33).Currently, it is believed that unlike most other CXC chemokines,g IP-10 and Mig have no activity on either neutrophils or mono-cytes, but appear to target stimulated lymphocytes specifically(34). The gene for CXCR3 is localized on human chromosomeXq13, which is in clear contrast to all other chemokine receptorgenes, suggesting a unique function(s) for this receptor and itsligands that may lie beyond their established role in T cell-depen-dent immunity (29). To date, there is no evidence in the literaturethatg IP-10 and/or Mig are necessary for recruitment or activationof eosinophils in vivo. There is more convincing evidence for IL-5as well as eotaxin in many allergic diseases with predominant eo-sinophilia. Our results have shown thatg IP-10 and Mig induceeosinophil chemotaxis via CXCR3 expression, and thatg IP-10-and Mig-induced eosinophil chemotaxis are up- and down-regu-lated by IL-2 and IL-10, respectively. Moreover, we have foundthat CXCR3 mRNA expression in eosinophils is up- and down-regulated by IL-2 and IL-10, indicating that IL-2 and IL-10 controlg IP-10- and Mig-induced eosinophil chemotaxis via regulation ofCXCR3 expression. Besides chemotaxis, we have found other bi-ological functions ofg IP-10 and Mig on eosinophils, includingECP release. To our knowledge, this is the first report that CXCR3is expressed on human peripheral eosinophils and that its expres-sion is regulated by T lymphocyte-associated cytokines. Thus, ourresults raise a conceptual issue of the potential biological or patho-physiological relevance of the presence of CXCR3 in humaneosinophils.

ECP, with storage and secreted forms (35), has a variety ofbiological activities, interacting with other immune cells andplasma proteins such as coagulation factors and proteins of thecomplement system. It is a major basic granule protein involved in

contact-dependent Ab-mediated cytotoxicity (36). ECP has beenused extensively as a marker for activation and secretion in eosin-ophils (37). A number of studies have documented that local andsystemic ECP measurement is a useful way to monitor eosinophilnumber and its activation in subjects with allergic disorders (38,39). In the present study we have demonstrated thatg IP-10 andMig activate eosinophils to release ECP. To our knowledge, this isthe first direct evidence of ECP release from eosinophils inducedby CXC chemokines. Our data demonstrate the ECP-releasing ca-pacity of both CXC chemokines, e.g.,g IP-10 and Mig, and CCchemokine, e.g., eotaxin. A rather complex picture is now begin-ning to take shape of how eosinophils selectively enter allergicinflammation sites and cause contact-dependent Ab-mediated cy-totoxicity by means of ECP release under association of chemo-kines and cytokines.

IL-10, an immunosuppressive and anti-inflammatory cytokineproduced by monocytes and T lymphocytes, regulates both inflam-matory/immune responses by not only modulating the activities ofT lymphocyte, B lymphocyte, and mononuclear phagocyte func-tion, but also by modulating polymorphonuclear cell-associatedchemokine expression (40). It has been reported that the newlydescribed CC chemokine HCC-4 is uniquely up-regulated byIL-10 (41), whereas another novel CC chemokine, designated al-ternative macrophage activation-associated CC-chemokine-1, isspecifically induced by IL-10 (42). On the other hand, IL-10 in-hibits the expression of IL-8, macrophage inflammatory protein-1a, macrophage inflammatory protein-1b, and KC in monocytesand macrophages (43–45). IL-10 preincubation resulted in the in-hibition of gene expression for several IFN-induced genes, such asg IP-10 and ICAM-1. The reduction in gene expression resultedfrom the ability of IL-10 to suppress IFN-induced assembly ofSTAT factors to specific promoter motifs on IFN-a- and IFN-g-inducible genes. IL-10 can directly inhibit STAT-dependent earlyresponse gene expression induced by both IFN-a and IFN-g inmonocytes by suppressing the tyrosine phosphorylation of STAT1(46). IL-10 might act indirectly to suppressg IP-10 expression byinhibiting LPS-induced class I IFN production (47). IL-10 selec-tively up-regulates the expression of CCR1, CCR2, and CCR5 inhuman monocytes by prolonging their mRNA half-lives, increas-ing the number of cell surface receptors, and producing a betterchemotactic responsiveness to relevant ligands (48). CXCR3 isexpressed preferentially in Th1 cells and in lymphoid organs of theIL-102/2 mouse, which develops chronic colitis (49). The partic-ipation of eosinophils in inflammation has often been linked toinflammatory responses with a so-called Th2 profile, such as var-ious forms of allergic or parasitic diseases. Our findings fit wellwith the evolving hypotheses that link chemokine ligand and re-ceptor pairs to Th1, Th2, or other types of inflammation and sug-gest that the eosinophil-mediated inflammation may not exclu-sively belong to Th2-type patterns. If stimulated by the Th1-typecytokine IL-2, CXCR3 is up-regulated, and the cell becomes re-sponsive to IFN-g-induced chemokines. On the other hand, if ex-posed to IL-10, this pathway is inhibited both by reduction ofIFN-g induced cytokines and by the expression of their receptor,CXCR3. Interestingly, the chemotactic activity of eotaxin, whichis believed to be mediated via CCR3, could not be up- or down-regulated by addition of a number of cytokines (Table I), suggest-ing that the eotaxin-CCR3 ligand-receptor pair is a constitutivechemotactic pathway for the eosinophils. Thus, IL-10 seems tosuppress two different steps in the inflammatory response mediatedby CXC chemokines acting via CXCR3. This is in concordancewith our demonstration that IL-10 can dramatically down-regulateCXCR3 expression on eosinophils as well as eosinophil chemo-taxis towardg IP-10 and Mig.

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Our data have pointed out an interesting phenomenon thatCXCR3 and its ligandsg IP-10 and Mig may play an importantrole in terms of eosinophil activation and trafficking during theallergic inflammation. However, there is no direct evidence of thesignificance of CXCR3 and its ligandsg IP-10 and Mig in vivoand/or pathophysiologically due to limited observation. A recentstudy showed that depletion/neutralization of eotaxin and/or IL-5in mice is sufficient to abolish eosinophilia in lung tissue and bron-choalveolar lavage fluid (50). These results emphasized the im-portance of the CCR3-eotaxin receptor-ligand pair in eosinophilrecruitment. They also raise an argument against an important rolein vivo for the CXCR3-mediated recruitment of eosinophils.Therefore, verifying and clarifying the significance of CXCR3 andits ligandsg IP-10 and Mig in vivo in humans in terms of themechanism of allergic inflammation will be interesting to examinein further studies.

In summary, we have documented that CXCR3 is expressed oneosinophils and is up- and down-regulated by IL-2 and IL-10, andthatg IP-10 and Mig, via CXCR3, activate eosinophils to chemo-taxis, ECP release, and NF-AT complex nuclear translocation. Thepresent study provides useful insights into a novel mechanism ofthe actions ofg IP-10 and Mig, which may be especially importantin the cytokine/chemokine environment for the pathophysiologicevents of allergic inflammation, including initiation, progression,and termination of the processes.

AcknowledgmentsWe thank Ulla Minura and Lisbethe Abrahamsen for their excellent tech-nical assistance. We express great gratitude to Lars Terenius, GeorgyBakalkin, and Tatjana Yakovleva (Department of Drug Dependence Re-search, Karolinska Institute, Stockholm, Sweden) for their generouscollaborations.

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