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Immobilized CXCR3 LigandsPlasmacytoid Dendritic Cell Recruitment by

Antal Rot and Dieter MaurerKriehuber, Peter Petzelbauer, Sabine Brandt, Georg Stingl, Norbert Kohrgruber, Marion Gröger, Paul Meraner, Ernst

http://www.jimmunol.org/content/173/11/6592doi: 10.4049/jimmunol.173.11.6592

2004; 173:6592-6602; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Plasmacytoid Dendritic Cell Recruitment by ImmobilizedCXCR3 Ligands1

Norbert Kohrgruber,* Marion Groger,† Paul Meraner,* Ernst Kriehuber,*‡ Peter Petzelbauer,†

Sabine Brandt,* Georg Stingl,* Antal Rot,§ and Dieter Maurer2*‡

Plasmacytoid dendritic cells (pDCs) recognize microbes, viruses in particular, and provide unique means of innate defense againstthem. The mechanism of pDC tissue recruitment remained enigmatic because the ligands of CXCR3, the cardinal chemokinereceptor on pDCs, have failed to induce in vitro chemotaxis of pDCs in the absence of additional chemokines. In this study, wedemonstrate that CXCR3 is sufficient to induce pDC migration, however, by a migratory mechanism that amalgamates thefeatures of haptotaxis and chemorepulsion. To mediate “haptorepulsion” of pDCs, CXCR3 requires the encounter of its cognateligands immobilized, optimally by heparan sulfate, in a form of a negative gradient. This is the first report of the absoluterequirement of chemokine immobilization and presentation for its in vitro promigratory activity. The paradigmatic example ofpDC haptorepulsion described here may represent a new pathophysiologically relevant migratory mechanism potentially used byother cells in response to other chemokines. The Journal of Immunology, 2004, 173: 6592–6602.

P lasmacytoid dendritic cells (pDCs)3 are professional APCswith the inherent capacity to produce large amounts ofnatural IFNs (nIFNs). pDCs are activated by viral patho-

gens which, in contrast, induce “paralysis” or apoptosis of con-ventional myeloid DCs (1–5). For example, HSV promotes sur-vival, evokes T cell stimulatory potential, and induces high levelnIFN production of pDCs (6–8). Consequently, pDCs can conferimmediate innate protection against viral replication (7). Unlikesecondary lymphoid tissues, the noninflamed organs are devoid ofpDCs. Thus, pDCs must home to virus-infected tissues to executethe first line innate defense against viral infections and, later, toserve as APCs for the induction of cellular antiviral immunity.However, up to now, the molecular mechanisms of pDC hominginto the sites of viral replication remain enigmatic.

Functionally different DC precursors have distinctive patterns ofchemokine receptor expression which determine their migratoryresponses. Epitheliotropic DC precursors respond to the CCR6 li-gand CCL20 (9, 10), precursors of the dermal/interstitial-type DClineage respond to CCR1/CCR5 and CCR2 ligands (9–11), andpDC precursors/immature pDCs express CXCR4 and high levels

of CXCR3, a receptor also present on Th1 and CTLs (6, 12–14).Accordingly, it has been speculated that L-selectin� pDCs useCXCR3 to access T cell-rich areas of inflamed lymph nodes (6).CXCR3 may also be the receptor guiding pDCs into virus-infectedtissue, as its three ligands CXCL9, CXCL10, and CXCL11 areinduced by IFN and are released rapidly after virus exposure (15–18). However, CXCR3 on pDCs apparently does not mediate che-motaxis in vitro (12, 14, 19). This does not reflect an indigenousinability of pDCs to chemotax because CXCL12 can induce mi-gration of these cells. As CXCL12 is a chemokine with constitu-tive expression in many tissues (9, 20, 21), it is unlikely that it isalone responsible for pDC migration into sites of viral infection.Accordingly, it has been shown that CXCR4-mediated migrationof pDCs is enhanced by CXCR3 ligand (CXCR3L) priming (14).This suggests that CXCR3 on pDCs functions solely as an ampli-fier of CXCR4-mediated chemotaxis. Thus, it is believed thatpDCs require the combined presence and action of CXCR3 and ofCXCR4 ligands to migrate into infected sites. These conclusionsare based on data obtained in Boyden-type chamber assays thatmeasure response to soluble chemokine gradients. It remained un-known whether CXCR3 alone could induce pDC migration inphysiologically more relevant systems such as adhesion to andmigration across endothelial cells (ECs). In this study, we demon-strate a novel mechanism of CXCR3-dependent pDC migrationthat does not involve CXCR4-mediated chemotaxis but requiresimmobilization and presentation of CXCR3Ls.

Materials and MethodsAbs and reagents

Purified nonlabeled mAbs were anti-CD3, anti-CD11b, anti-CD16, anti-CD19, anti-CD34, anti-CD41, anti-CD56, anti-CD235a (Immunotech,Marseille, France); anti-CCR3, anti-CXCL9, anti-CXCL10, anti-CXCL11,anti-CXCL12 (R&D Systems, McKinley Place, MN); and anti-CD8 andanti-CD45RA (both from BD Biosciences, Mountain View, CA). HECA-452 was kindly provided by Dr. L. Picker (University of Texas Southwest-ern Medical Center, Dallas, TX). PE-conjugated mAbs were anti-CD123,anti-CXCR1, anti-CXCR2, anti-CXCR4, anti-�7-integrin, anti-CD104 (BDPharmingen, San Diego, CA), anti-CCR7, anti-CD4, anti-L-selectin (BDBiosciences), anti-CCR2, anti-CCR6, and anti-CXCR5 (R&D Systems).FITC-conjugated mAbs were anti-CCR5, anti-CXCR3 (R&D Systems),

*Division of Immunology, Allergy and Infectious Diseases and †Division of GeneralDermatology, Department of Dermatology, Medical University of Vienna, ‡Center ofMolecular Medicine (CeMM) of the Austrian Academy of Sciences, and §NovartisInstitute for Biomedical Research, Vienna, Austria

Received for publication July 15, 2004. Accepted for publication September 2, 2004.

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 CeMM of the Austrian Academy of Sciences, theAustrian Science Foundation (Fonds zur Forderung der Wissenschaftlichen For-schung; Project SFB F018/13), and the Genome Research Programme Austria (GEN-AU) of the Austrian Ministry of Science (to D.M.).2 Address correspondence and reprint requests to Dr. Dieter Maurer, CeMM andDepartment of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail address: dieter.maurer@akh-wien.ac.at3 Abbreviations used in this paper: pDC, plasmacytoid dendritic cell; nIFN, naturalIFN; CXCR3L, CXC chemokine receptor 3 ligand; EC, endothelial cell; FAK, focaladhesion kinase; TRITC, tetramethyl isothiocyanate; HS, heparan sulfate; CS, chon-droitin sulfate; PTX, pertussis toxin; DMEC, dermal microvascular EC; RT, reversetranscriptase; LSM, laser scanning microscopy; TEM, transendothelial migration;PMN, polymorphonuclear neutrophil; PSGL, P-selectin ligand; CLA, cutaneous lym-phocyte Ag; pFAK, phosphorylated FAK.

The Journal of Immunology

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and anti-CD45RA (BD Biosciences). Biotinylated anti-CCR1 and anti-CD123 were obtained from R&D Systems and BD Pharmingen, respec-tively. PerCP-conjugated anti-HLA-DR was obtained from BD Bio-sciences. HRP-conjugated phosphotyrosine-specific (PY-20) and focaladhesion kinase (FAK) phosphorylation site (Y397)-specific mAbs wereobtained from BD Transduction Laboratories (San Jose, CA). Second stepAbs included Alexa 488- and tetramethyl isothiocyanate (TRITC)-labeledgoat anti-rabbit IgG (Molecular Probes, Leiden, The Netherlands) andTRITC- and Cy5-labeled goat F(ab�)2 anti-mouse IgG (Jackson Immu-noResearch Laboratories, West Grove, PA). Lymphatic vessel-specific anti-podoplanin Abs were generated by repeated immunizations of rabbitswith the extracellular domain of human podoplanin expressed in Esche-richia coli.

Recombinant human chemokines used included CCL2, CCL3, CCL5,CCL7, CCL8, CCL13, CCL17, CCL19, CCL20, CCL22, CXCL8,CXCL11, CX3CL1 (R&D Systems), CCL22, CCL25, CXCL9, CXCL10,and CXCL12 (Strathmann Biotec, Hamburg, Germany). Human rTNF�and rIL-3 were obtained from Strathmann Biotec. IFN-�2b (intron-A) wasobtained from Schering-Plough (Kenilworth, NJ). Heparan sulfate (HS)and chondroitin sulfate (CS) were obtained from Sigma-Aldrich (St. Louis,MO) and heparitinase II and chondroitinase ABC from Seikagaku (Tokyo,Japan). Wild-type HSV-2 and influenza virus strain A/Puerto Rico/8/34/H1N1 were kindly provided by Drs. H. Hofmann (Department of ClinicalVirology, Medical University of Vienna, Vienna, Austria) and T. Muster(Department of Dermatology, Medical University of Vienna, Vienna, Aus-tria), respectively. Pertussis toxin (PTX) was from Calbiochem (Darmstadt,Germany).

Cell preparations

pDCs were purified from the peripheral blood of healthy donors essentiallyas described (22). Briefly, PBMCs were isolated by Ficoll-Hypaque (Phar-macia, La Jolla, CA) density gradient centrifugation and sheep RBC-bind-ing T cells were removed. The resulting cell fraction was then depleted ofresidual T, B, NK, and hemopoietic stem cells, monocytes, basophils,platelets, and erythrocytes by anti-CD3/CD11b/CD16/CD19/CD34/CD41/CD56/CD235a (2 �g/ml each) immunolabeling and anti-mouse IgG im-munomagnetic depletion (MACS; Miltenyi Biotec, Auburn, CA). Sixty to85% of the remaining cell fraction qualified as pDCs by immunopheno-type, i.e., CD123�CD45RA�HLA-DR�CD11c�, as revealed by FACSanalysis (FACScan; BD Biosciences). T cells were obtained from the sheepRBC-bound PBMC fraction by hypotonic lysis. Polymorphonuclear neu-trophil (PMN) cells were isolated by centrifugation of heparinized bloodover a continuous isomolar 70% Percoll gradient (Amersham Biosciences,Arlington Heights, IL) as described previously (23). Human umbilical veinECs and dermal microvascular ECs (DMECs) were isolated and propa-gated as described elsewhere (24, 25).

RNA extraction and RT-PCR

Total RNA was isolated from pelleted DMECs and flow-sorted (FACStarPLUS;BD Biosciences) CD123�HLA-DR�CD11c� pDCs (purity �99%) usingTriPure reagent according to the manufacturer’s instructions (Roche Di-agnostics, Basel, Switzerland). DNase 1 digestion and enzyme inactivationwere performed in RNeasy microcolums (Qiagen, Valencia, CA) as rec-ommended. DNA-free DMEC and pDC RNAs were eluted in 20 �l ofRNase-free water and cDNA synthesis was conducted in M�lTI Ultra PCRtubes (Sorenson Bioscience, Salt Lake City, UT), each containing 50 mMTris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 2 mM of eachdNTP, 200 pmol random hexamer (Roche Diagnostics), 20 U of recom-binant RNase inhibitor (RNaseOUT; Invitrogen Life Technologies, Carls-bad, CA), 9.5 �l of RNA and 200 U of SuperScript III reverse transcriptase(�RT; Invitrogen Life Technologies) or 1 �l of 87% glycerol as mock RTcontrol (�RT) in a total volume of 20 �l. After incubation for 1 h at 48°Cin a thermocycler, 2.5-�l aliquots of each reaction mixture were subjectedto PCR using two different primer combinations for specific amplificationof CXCR3 splice variants A and B (26). For amplification of a 281-bpfragment of variant A, primer 5� cxcr3 Aj (5�-ccatggtccttgag/gtgagtgacc-3�) including translation start and spanning the exon1/exon2 junction of theA variant was combined with primer 3� cxcr3 AB (5�-gagcaggaaggtgtcggtgctgc-3�) recognizing both variants. For amplification of a variant B-specific 449-bp fragment, primer 5� cxcr3 B (5�-gctgagcggatggagttgaggaag-3�) corresponding to exon 2 of variant B was used in combinationwith the consensus primer 3� cxcr3 AB. Fifty-microliter reaction mixtureswere prepared in M�lTI Ultra PCR tubes, each containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 1.5 mM of each dNTP, 100pmol primer 3� cxcr3 AB, 100 pmol primer 5� cxcr3 Aj, or 5� cxcr3 B,2.5-�l template, and two drops of mineral oil as top layer. After a hot startat 95°C for 5 min and addition of 1.5 U of Taq polymerase/tube, a PCR

program consisting of seven touch-down cycles (denaturation at 92°C for30 s/primer annealing at 72°C-63°C for 45 s (�1.5°C/cycle)/primer elon-gation at 72°C for 45 s) followed by 38 standard cycles (92°C for 30 s/63°Cfor 45 s/72°C for 45 s) was conducted in a MiniCycler MJ Research(Biozym, Oldendorf, Germany). Resulting amplification products were vi-sualized on a 1.5% Tris-acetate agarose gel by ethidium bromide staining.

Stimulation of pDCs and T cells

pDCs were seeded in 96-well flat-bottom microtiter plates (Costar, Cam-bridge, MA) in RPMI 1640 supplemented with 10% FCS, 2.5 mM L-glu-tamine, 100 U/ml penicillin, and 100 �g/ml streptomycin (all from Invitro-gen Life Technologies, Rockville, MD). pDCs were cultured in the absenceor presence of IL-3 and TNF-� (100 U/ml each), HSV-2 (1 � 106 PFU/ml),influenza virus (strain A/PR-8, 5 hemagglutination units/ml), or IFN-�(100 U/ml) for the indicated time periods. CXCR3-expressing T cells weregenerated by culture of purified T cells in the presence of IL-2 (100 U/ml)for 14 days as described previously (27).

Transwell insert migration and adhesion assays

Chemotaxis assays were performed as described previously (9). Briefly,pDCs either freshly isolated or stimulated as indicated were washed andresuspended in migration buffer (HBSS; Invitrogen Life Technologies), 1mM CaCl2, 0.5 mM MgCl2, 0.1% BSA (Sigma-Aldrich)) at a density of2–3 � 106 cells/ml. Chemokine solution or buffer alone was added toindividual wells of 24-well plates (Costar) on ice before Costar transwelldevices (5-�m pore size) were inserted into the wells. Suspended cellswere layered on top of the membrane and allowed to migrate for 3 h at37°C. Transmigrated cells were recovered from the fluid phase of the lowerwell, stained with anti-CD123-PE, anti-CD45RA-FITC, and anti-HLA-DRPerCP simultaneously, and pDCs were enumerated by FACS analysis asdescribed (9).

In a different set of experiments, we assessed migration in response tochemokines immobilized onto transwell filters. Chemokines (1–100 ng/ml)were allowed to bind to the upper, to the lower, or simultaneously to theupper and lower side of HS or CS (both 100 �g/ml)-coated or noncoatedfilters for 20 min at room temperature. After washing the filter insert, cellswere added into the upper well and allowed to migrate for 3 h at 37°C.Then, membranes were cut out, stringently washed with ice-cold PBS, andadherent cells were fixed in ethanol/acetone (1:1) at �20°C for 15 min andstained with anti-CD123-PE (5 �g/ml). Migrated pDCs, i.e., CD123� cellsattached to the lower surface of the transwell membranes, were visualizedby laser scanning microscopy ((LSM) LSM 510; Zeiss, Oberkochen, Ger-many) and enumerated automatically by a previously described algorithmand NIH image software (23). Migration indices were obtained as a ratioof pDCs migrated in the presence of chemokine and pDCs migrated inresponse to buffer. In addition, pDCs were counted on the upper side of thetranswell filters to estimate their adhesion. Adherent pDCs were fixed with4% paraformaldehyde, permeabilized with 0.1% saponin in PBS/1% BSA,and stained with anti-phospho(p)FAK (1 �g/ml) followed by goat anti-mouse IgG-Alexa488 (2 �g/ml). After quenching in normal mouse serum,cells were incubated with TRITC-coupled phalloidin (5 �g/ml; Sigma-Aldrich) and anti-CD123-biotin (10 �g/ml) followed by streptavidin-Cy5(1 �g/ml; Molecular Probes). Control stainings were performed with ap-propriate isotype-matched Abs (2 �g/ml; Sigma-Aldrich).

Transendothelial migration (TEM) assays

TEM assays were performed as described (23). Briefly, ECs were seededon the top of a collagen gel (Vitrogen 100; Invitrogen Life Technologies)and cultured to confluence in IMDM (Invitrogen Life Technologies) sup-plemented with 20% FCS, EC growth supplement (Promo Cell, Heidel-berg, Germany), 2.5 mM L-glutamine, 100 U/ml penicillin, and 100 �g/mlstreptomycin. After repeated washings with RPMI 1640, EC monolayerswere incubated with the indicated chemokine for 10 min at 37°C followedby multiple rounds of washes. pDCs, T cells, or PMN cells were loadedwith Cell Tracker Green (Molecular Probes), resuspended in RPMI1640/5% BSA (106 cells/ml), and seeded on top of the EC monolayer.Cells were then allowed to adhere to and migrate through the EC mono-layer for 3 h at 37°C. In chemokine prepulsing experiments, pDCs wereincubated with the indicated chemokine (1–100 ng/ml) for 20 min on ice,washed three times, and seeded onto the EC monolayer. Where indicated,pDCs were pretreated with 0.2 �g/ml PTX for 2 h before seeding themonto the EC monolayer. Migration was stopped by fixing the collagen gelswith 4% paraformaldehyde and pDCs were visualized by anti-CD123 (5�g/ml) and anti-mouse IgG-TRITC (2 �g/ml) immunostaining. Adherentand transmigrated cells were identified as Cell Tracker Green� cells (andCD123� cells in the case of pDCs) by confocal LSM and were enumeratedusing NIH image software as described (23).

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Tyrosine phosphorylation assay

Phosphotyrosine blotting was performed as described previously (28) withmodifications. Cell pellets were lysed in 1% Brij97 (Sigma-Aldrich) lysisbuffer (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 20 mM NaF (Sigma-Aldrich), 1 mM Na3VO4 (Calbiochem)) supplemented with a completeprotease inhibitor mixture (Roche Diagnostics). After 30 min at 4°C, ly-sates were centrifuged to remove nuclei at 15,000 � g for 2 min. Super-natants were mixed with an equal volume of 2� reducing sample bufferand boiled for 3 min. Isolated proteins were resolved by gradient (5–12%)SDS-PAGE and blotted onto polyvinylidene difluoride membranes. Mem-branes were blocked using a buffer containing 1% BSA, 20 mM Tris-HCl,pH 7.4, 150 mM NaCl, 0.1% Tween 20, 0.1 mM Na3VO4 and incubatedwith HRP-conjugated PY-20 mAb (0.125 �g/ml). Phosphotyrosine-modi-fied proteins were visualized using ECL Western blotting detection re-agents (Amersham Biosciences).

Chemokine binding to ECs

ECs were detached by trypsin/EDTA (Invitrogen Life Technologies),washed, and incubated with the indicated chemokines (1–1000 ng/ml) for20 min on ice. After several washes in ice-cold Ca2�/Mg2�-containingPBS, cells were exposed to rabbit anti-CXCL10 or anti-CXCL12 Abs (1�g/ml each) followed by Alexa 488-labeled anti-rabbit IgG (2 �g/ml).Where indicated, ECs were treated with 25 mU/ml heparitinase II or chon-droitinase ABC for 1 h at 37°C, washed three times, and then subjected tothe chemokine binding assay. Cell-bound Alexa 488 immunofluorescencewas analyzed on a FACScan.

Immunohistochemistry

All human tissue specimens were obtained upon informed consent. Piecesof normal human skin were taken during elective plastic surgery. Four-millimeter punch biopsies of lesional tissue were obtained from patientsaffected by HSV-2 or varicella zoster virus. Tonsils were removed in theprocess of elective tonsillectomy. All samples were snap-frozen in liquidnitrogen-chilled isopentane. Frozen sections were mounted onto glassslides and fixed with acetone at room temperature. For staining, the slideswere hydrated and incubated with mouse anti-CXCL9, anti-CXCL10, oranti-CXCL11 mAbs. The bound primary mAbs were detected by sequen-tial incubations with alkaline phosphatase-conjugated rabbit anti-mouseand an alkaline phosphatase-anti-alkaline phosphatase staining kit (DAKO,Carpinteria, CA) according to the manufacturer’s instructions. Isotype-matched control mAbs (DAKO) were used at equimolar concentrations.Each immunostaining protocol was performed on tissue samples from atleast three different donors.

Immunofluorescence analysis

Five-micrometer cryosections were mounted onto glass slides, air-dried,and then fixed with acetone for 20 min. After drying, slides were hydratedwith Ca2�/Mg2�-deficient PBS and exposed to anti-CD45RA or anti-CD8(1:50 in PBS/1% BSA) for 45 min at room temperature followed by wash-ings and incubation with Cy5-labeled goat F(ab�)2 anti-mouse IgG (5 �g/ml) for 45 min at room temperature. After blocking with 1% mouse serumin PBS/BSA, the sections were exposed sequentially to biotinylated anti-CD123 (10 �g/ml) and streptavidin-Oregon Green (1 �g/ml; MolecularProbes). After several washings, the sections were stained with PE-labeledanti-CD4 (1/50 diluted in PBS/BSA), mounted with Fluoprep (BioMerieux,Marcy l’Etoile, France), and examined by confocal LSM. To detect lymphaticvessels, sections were incubated with rabbit anti-podoplanin antiserum or pre-immune serum (final dilution: 1/2000) followed by TRITC-labeled goatF(ab�)2 anti-rabbit IgG. In these experiments, pDCs were simultaneously vi-sualized by anti-CD123 and anti-CD45RA immunostaining. To characterizechemokine receptor expression in tissue cells, double labeling experimentswere performed with FITC- or PE-labeled anti-chemokine receptor mAbs andbiotinylated anti-CD123 followed by TRITC- or Oregon Green-labeledstreptavidin.

ResultsCirculating pDCs express CXCR3 but fail to migrate to solublegradients of CXCR3 ligands

pDCs, identified as CD123�CD45RA� cells in lineage Ag-depleted PBMCs, express CXCR3 uniformly and at high levels(Table I). CXCR4 is expressed by pDCs but its expression variesbetween pDCs from different donors (range: 7–86% positivepDCs; Table I). CCRs 1 through 7 and CXCR1, CXCR2, andCXCR5 are not or are only weakly expressed by blood pDCs. The

majority of blood pDCs display substantial amounts of L-selectinwhich they lose during the process of purification (Table I). Inaddition, pDCs express high levels of the P-selectin ligand(PSGL)-1 and its fucosylated variant, the cutaneous lymphocyteAg (CLA; Table I). Furthermore, nearly all pDCs express �4�7

integrin, the receptor for the mucosal addressin cell adhesion mol-ecule. �E, �6, and �4 integrins were not detected on pDCs in sig-nificant levels (Table I). Thus, pDCs consistently express one pre-ponderant chemokine receptor, CXCR3, and display the relevantreceptors for homing into the skin, the intestine, and secondarylymphoid tissues. We next analyzed isolated pDCs for their abilityto migrate to a variety of chemokines in a transwell insert chemo-taxis assay. pDCs did not transmigrate through the filters in re-sponse to CXCR3Ls irrespective of the chemokine concentrationused (Fig. 1) but responded to CXCL12 (Fig. 1A). Conversely,IL-2-activated CXCR3� T cells were vigorously attracted by sol-uble CXCL10 gradients (Fig. 1B). pDCs were not significantlyattracted by any of the other chemokines tested (Fig. 1A). pDCsmatured in vitro in the presence of IL-3 and TNF-� migrated ef-ficaciously to the CCR7 ligand CCL19 but not to CXCR3Ls (Fig.1B, see below) or to any other chemokines tested (data not shown).These results closely mirror previously published results obtainedin Boyden-type chamber assays (12–14).

We next asked whether the apparent nonresponsiveness of pDCsin Boyden-type chamber assays is due to the expression of a mi-gration-incompetent form of CXCR3. Indeed, recent data indicatethat two splice variants of CXCR3, CXCR3A and CXCR3B, exist(26). While CXCR3A mediates chemotaxis, CXCR3B may be in-volved in transmitting angiostatic signals to ECs. As revealed by asensitive and splice variant-specific RT-PCR assay, pDCs express

Table I. Chemokine receptor and adhesion molecule expression onpDCsa

Surface ReceptorExpression Intensity

(MFI)bPositive Cells

(%)c

CCR1 1 �1 14 �8CCR2 3 �1 32 �21CCR3 �1 �1CCR5 7 �4 19 �16CCR6 �1 �1CCR7 2 �1 9 �3CXCR1 �1 �1 �1CXCR2 �1 �1 �1CXCR3 25 �1 97 �2CXCR4 5 �5 47 �40CXCR5 �1 �1L-selectind 3 �3 3 �2PSGL-1 578 �224 98 �2CLA 190 �107 98 �1�4 integrin 281 �81 99 �0�7 integrin 21 �7 79 �16�E integrin 1 �1 5 �3�6 integrin 2 �3 11 �17�4 integrin �1 �1

a pDCs were identified as CD123�CD45RA� cells in PBMC fractions depleted oflineage Ag-expressing cells and analyzed for chemokine receptor and adhesion mol-ecule expression by mAb labeling and FACS.

b pDC mAb reactivities given were calculated by subtracting the fluorescenceintensity (MFI) values produced by control mAbs from the MFI values produced bythe specific mAbs.

c Percentage of pDCs reactive with mAbs recognizing the indicated moieties.d Anti-L-selectin reactivity of pDCs gated as CD123�CD45RA� cells was also

assessed in PBMC preparations before depletion of lineage Ag-expressing cells. Inpre-enrichment samples, L-selectin was vigorously expressed by pDCs (MFI: 386 �76; percent-positive pDCs: 92 � 4), indicative of L-selectin shedding during thepurification procedure. No significant expression difference between pre- and posten-richment was noted for the other surface molecules investigated. Data are expressedas mean values (�SD) obtained in five independent experiments.

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CXCR3A but fail to display CXCR3B-encoding RNA (Fig. 2). Incontrast, human microvascular ECs are positive for CXCR3B (Fig.2). This indicates that pDCs are, in principle, equipped with thepotential to migrate in response to CXCR3 ligands.

Apically immobilized CXCR3 ligands induce transmigrationof pDCs

It has been suggested that immobilized chemokines induce leuko-cyte adhesion to ECs and consequent TEM in vivo (29). Hapto-taxis, i.e., migration along a positive gradient of immobilized che-moattractant, was shown to be the mechanism of neutrophil andmonocyte migration (30). To investigate the possible dependencyof pDC migration on chemokine immobilization, we precoated5-�m pore-size transwell filters on the apical, on the basal, or onboth the apical and the basal side with CXCL10. The coating con-ditions used allow for the immobilization of significant levels ofchemokine on the selected side of the filter (A. Rot, unpublishedobservations). Purified pDCs were placed into the upper chamberof the transwells and the cells migrated were enumerated on thebasal side of the filter and in the lower chamber. pDCs transmi-

grated when CXCL10 was immobilized onto the upper side of thefilter but no response was observed when the chemokine was im-mobilized on the lower side or on both sides of the membrane (Fig.3A). In contrast, CXCL10 immobilized on the basal filter side pro-moted weak T cell migration (Fig. 3A). No T cell migration wasinduced when CXCL10 was immobilized on the apical filter side(Fig. 3A). In none of the experimental setups above were pDCs orT cells detected in the fluid phase or adherent to the bottom of thelower chamber (data not shown). Thus, pDCs can sense a negativestep gradient of immobilized CXCL10 and migrate in response bya mechanism that entails features of haptotaxis (i.e., dependencyon chemokine immobilization) and chemorepulsion (i.e., move-ment away from highest chemokine concentration).

HS saccharides that decorate various proteins are believed tobind and present chemokines in vivo, in particular, on the apicalEC surface (29–32). To deconstruct such a chemokine presenta-tion setup, the top sides of filters were coated with HS or CS beforepulsing them with CXCL10 and measuring pDC migration in re-sponse. As shown in Fig. 3B, HS but not CS enhanced CXCL10-mediated pDC migration. HS-bound CXCL10 more efficaciouslyinduced pDC migration than polycarbonate-bound CXCL10 (Fig.3B). Binding studies with titrated amounts of radiolabeledCXCL10 showed that, upon exposure to suboptimal chemokineconcentrations, the HS-coated filters bound up to 5-fold more che-mokine than nonmodified filters (data not shown). For control pur-poses, HS, CS or mixtures of CXCL10 and HS or CS were placedin soluble form into the lower chamber of the transwells. None ofthese experimental conditions resulted in migratory responsescomparable to those induced by HS-immobilized CXCL10 (Fig.3B). To see whether pDC migration in response to HS-boundCXCL10 is restricted to apically bound chemokine, HS coatingwas implemented in an experimental setup analogous to Fig. 3A.As shown in Fig. 3C, pDCs transmigrated when HS/CXCL10 wasimmobilized onto the upper side of the filter but no response wasobserved when HS/CXCL10 was immobilized on the lower side oron both sides of the membrane. Thus, CXCL10 immobilized andpresented apically in a physiologically relevant fashion is sufficientto induce pDC migration.

Induction of pDC adhesion, morphological changes, andtyrosine phosphorylation by immobilized CXCL10

The following assays were performed to study the outcome of pDCstimulation by immobilized CXCL10. As shown in Fig. 4A, pDCsstrongly adhered to HS/CXCL10-bound, but not to HS-coated,membranes. In contrast, exposure of pDCs to soluble CXCL10 didnot induce adhesion to the substratum (data not shown). Also, only

FIGURE 1. Soluble CXCL10 gradients do notinduce transmigration of pDCs. A, Migration ofpDCs to soluble chemokine gradients. The indi-cated chemokines (100 ng/ml each) were added tothe lower chamber of transwells. pDCs were al-lowed to migrate through 5-�m pore-size filtersfor 3 h. Migrated pDCs, i.e., CD123�CD45RA�

HLA-DR� cells, were enumerated in the fluidphase of the lower chamber by FACS analysis.Mean migration indices (�SD, n � 5 experi-ments) are given on the x-axis. B, T cells but notpDCs migrate to soluble CXCL10 gradients. IL-2-stimulated CXCR3� T cells (�), immaturepDCs (f), or IL-3/TNF-�-activated pDCs (u)were allowed to migrate to titrated concentrationsof soluble CXCL10 (y-axis, 1–1000 ng/ml). Mi-gration indices obtained in one experiment repre-sentative of three are given on the x-axis.

FIGURE 2. pDCs express transcripts coding for the A but not the Bform of CXCR3. DNA-free RNA was extracted from FACS-purified pDCsand cultured DMECs. cDNA (�RT) and the corresponding RNA template(�RT) were subjected to CXCR3A- and CXCR3B-specific PCR amplifi-cation. pDCs express RNA coding for CXCR3A (281-bp amplicon) selec-tively while DMECs display CXCR3A and CXCR3B (449-bp amplicon)-specific transcripts. No amplicons were observed when the RT reaction wasomitted, excluding the possibility that the non-intron-spanning primer pairfor CXCR3B amplification produced positive results due to genomic DNAcontamination of the DMEC sample. DNA molecular mass marker (M) and16-�l aliquots of each reaction were run on agarose gels and visualized byethidium bromide staining. Positions of marker bands and positions ofpredicted CXCR3A and CXCR3B amplicons are indicated at the left andright margins, respectively.

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immobilized but not soluble CXCL10 induced intracellular signal-ing as evidenced by enhanced tyrosine phosphorylation of severalcytosolic pDC proteins with a molecular mass of �120–140 kDa(Fig. 4B, left panel). In contrast to pDCs, T cells failed to phos-phorylate cellular proteins when exposed to HS-immobilized che-mokines (Fig. 4B, right panel).

pDCs that migrated in response to HS-immobilized CXCL10formed lamellipodia and long “dendrites” that impinge through thepores of the membrane (Fig. 4C, left panels). These morphologicalterations were not seen in the few cells that adhered and migratedspontaneously in the absence of chemokine (Fig. 4C, right panels).Confocal LSM of pDCs during the process of transmigration re-vealed clustering of phosphorylated FAK (pFAK) as well as ofIL-3R�/CD123 next to the edges of the filter pores (Fig. 4D). Asexpected, pan-FAK immunostaining also revealed a redistributionof pDC-expressed total FAK toward the edges of the pores andpFAK clustering occurred only when filters were coated withCXCL10/HS but not when the filters were coated with HS only(data not shown). This suggests that pDCs sense the immobilizednegative step gradient of CXCL10 in or around the pores and re-spond by adhesion and migration.

CXCL10 and CXCL12 are presented by HS moieties on ECsurfaces

Next we investigated whether CXCL10 and CXCL12, the two pro-totypic chemokines that stimulate pDCs, can bind to ECs in vitro.ECs were pulsed with CXCL10 and CXCL12 and chemokines

bound were detected by specific Abs and FACS. While nontreatedECs were devoid of CXCL10 and CXCL12, chemokine pulsing ofECs resulted in the appearance of significant surface immunore-activity (Fig. 5A). No significant CXCL10 and CXCL12 bindingwas observed to heparitinase-treated ECs (Fig. 5A). Control chon-droitinase treatment did not significantly affect chemokine bindingto ECs (data not shown). Moreover, CXCL10 and CXCL12 cross-competed for EC binding (Fig. 5A) indicating that ECs display alimited number of moieties capable of HS-dependent chemokinebinding. In binding studies with titrated concentrations of CXCL10we observed that CXCL10 binding to ECs was saturable and op-timal at rather low chemokine concentrations of around 100 ng/ml(Fig. 5B). A similar dose-binding relation was seen with CXCL12(data not shown).

To explore whether EC-associated CXCR3Ls can be detected invivo, we studied HSV-infected skin by immunohistochemistry. Allthree CXCR3Ls were found to be present in the lesions and weredetected in the epidermis most prominently (Fig. 5C). CXCL10and CXCL11 were further found associated with dermal micro-vascular ECs (insets of Fig. 5C; lower left panel and upper rightpanel, respectively). Staining of normal human skin withCXCR3L-specific mAbs and isotype control staining of lesionalskin revealed no immunoreactivity (Fig. 5C, data not shown).

EC-bound CXCL10 induces adhesion and migration of pDCs

To further explore the biologic relevance of chemokine presentation-dependent pDC migration, we asked whether EC-bound chemokines

FIGURE 3. Apically immobilized CXCR3 ligands induce pDC migration. A, CXCL10 immobilized on the upper surface but not CXCL10 immobilizedon the lower surface or on both sides of the transwell filter induces pDC transmigration. CXCL10 (100 ng/ml) was coated onto the upper and/or the lowersurface of the polycarbonate transwell filter. Then, pDCs or T cells were placed in the upper chamber of the transwell and, after 3 h of incubation, filterswere washed stringently. Filter-bound cells were fixed and stained with anti-CD123 and anti-CD3 mAbs for the detection of pDCs and T cells, respectively.Migrated pDCs or T cells on the lower side of the filter were visualized by confocal LSM and enumerated by automatic cell counting. Mean migrationindices (�SD) of pDCs (f) and T cells (�) as obtained in duplicate experiments and counting of 10 microscopic high power fields (HPF) per experimentalcondition are given on the x-axis. Mean baseline migration of pDCs and of T cells corresponds to 49 and 10 cells/HPF (0.01 mm2), respectively. Resultsare representative for three independent experiments. �, Significant (p � 0.01, Student’s t test) increase in migration as compared with respective mediumcontrol. B, HS but not CS enhances CXCL10 immobilization-dependent pDC transmigration. Transwell filters were coated on the upper side with CS orHS (100 �g/ml each), washed, and then pulsed with CXCL10 (100 ng/ml) or left untreated (f). As a control, CXCL10 (100 ng/ml), CS, HS, (100 �g/mleach), or mixtures thereof were placed into the lower chamber of the transwells in soluble form (�). pDCs were added to the upper chambers of thetranswells and pDC migration evaluated as described. Results are representative for three independent experiments. �, Significant (p � 0.01) increase inmigration as compared with respective medium control; ��, significant (p � 0.01) increase in migration of pDC across HS � CXCL10-coated membranesas compared with pDC migration across CXCL10-coated filters. C, HS/CXCL10 immobilized on the upper surface but not HS/CXCL10 immobilized onthe lower surface or on both sides of the transwell filter induces pDC transmigration. HS was coated onto the upper and/or the lower surface of the filterand filters were incubated with CXCL10 at the side of previous HS exposure. pDC transmigration was assessed and data are presented as in A. �, Significant(p � 0.01) difference in migration.

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can also provide the essential stimulus for pDC transmigrationthrough EC monolayers. Resting and TNF-�-activated EC monolay-ers grown on collagen gels were pulsed with CXCL10. pDCs wereplaced apically onto ECs and adhesion and TEM were assessed bymultilevel confocal LSM. In accordance with the results obtained inthe transwell assays, ECs supported significant CXCL10-dependentadhesion (Fig. 6A) and TEM (Fig. 6B) of pDCs. EC-bound CXCL12also promoted adhesion (Fig. 6A) and TEM (Fig. 6B) of pDCs, thoughmostly to a lesser extent than CXCL10. Resting ECs supported pDCadhesion and migration to CXCL10 more potently than activated ECs(Fig. 6, A and B). In the same experiment, selectively activated ECsbut not resting ECs promoted the transmigration of PMN (Fig. 6C). Inagreement with the results of the transwell experiments, activated Tcells showed only inefficient migration through EC monolayers pre-senting CXCL10 apically (Fig. 6D).

CXCR3-dependent pDC migration across EC monolayers isinhibited by soluble chemokines and PTX

So far we have shown that soluble CXCR3Ls have no promigra-tory effect on pDCs. We asked whether soluble CXCR3Ls canaffect the TEM induced by immobilized chemokines. This is arelevant question because under pathologic conditions CXCR3Ls

can be present in the circulation (33, 34). pDCs were pretreatedwith CXCL9, CXCL10, or CXCL11 before placing them over ECmonolayers presenting immobilized CXCL10. Pretreatment witheach of the CXCR3Ls abolished the adhesion of pDCs to ECs andtheir TEM (Fig. 7A). Inhibition of pDC migration was, however,not ligand specific: pretreatment of pDCs with either CXCR3Ls orCXCR4Ls resulted in the inhibition of both their adhesion andmigratory responses to either of these ligands immobilized on theEC surface (Fig. 7B). It further appears that the signal transductionrequired for CXCL10-mediated pDC adhesion and TEM is differ-ent. Adhesion is insensitive to PTX (Fig. 7C), while transmigrationis abolished by Gi-protein blockade (Fig. 7D).

CXCR3-positive pDCs and T cells accumulate in herpesvirus-induced skin lesions

Next we investigated whether pDCs infiltrate virus-induced skin erup-tions. pDCs can be reliably identified by their CD4�CD45RA�

CD123bright immunophenotype (35–37). We found pDCs localized inperivascular clusters in HSV (Fig. 8, A and B; pDCs appear aswhitish and yellow cells, respectively) and in varicella zoster vi-rus-induced skin eruptions (data not shown). Clusters of pDCswere surrounded by a mixed infiltrate consisting of CD4� and

FIGURE 4. Immobilized CXCL10 induces adhesion, tyrosine phosphorylation, and polarization of pDCs. A, HS-bound CXCL10 mediates pDC adhe-sion. Transwell filters were coated with HS only or sequentially with HS and CXCL10. pDCs were allowed to adhere to the filters for 20 min, andfilter-bound cells were stained with anti-CD123 mAbs and visualized by confocal LSM (upper panels). Mean numbers (�SD) of filter-bound pDCs wereassessed by automated cell counting (lower panel, y-axis). �, Significant (p � 0.01) increase in adhesion. B, Immobilized but not soluble CXCL10 inducestyrosine phosphorylation in pDCs as assessed by anti-phosphotyrosine Western blotting. pDCs (left panel) or IL-2-stimulated T cells (right panel) wereincubated in the absence or presence of soluble CXCL10 (100 ng/ml) or were spun down gently onto HS only or HS/CXCL10-coated filters and culturedfor 10 min. Solid arrowhead denotes the position of cellular proteins that are hyperphosphorylated in pDCs in response to immobilized CXCL10. Openarrowhead points to protein bands hyperphosphorylated in T cells in response to soluble but not immobilized CXCL10. C, Morphology of pDCs adheringand transmigrating in response to immobilized CXCL10. Flattening and lamellipodium-like protrusions are seen in pDCs adhering to HS/CXCL10 (upperleft panel) but not to HS-coated filters (upper right panel). pDCs migrated to immobilized CXCL10 develop dendrite-like protrusions (lower left panel).No substantial pDC migration was observed on HS-coated filters (lower right panel). Fixed cells were visualized by propidium iodide staining and confocalLSM. D, pDCs cluster pFAK next to the pores of HS/CXCL10-coated filter membranes. Confocal LSM of pDCs adhering to HS/CXCL10-coated filtersvisualizes the distribution of pFAK (green), F-actin (red), and CD123 (blue; overlay image in lower right panel). The other three panels give the imagesof the individual fluorescence channels.

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CD8� T cells (Fig. 8, A and B). Normal skin did not contain pDCs(not shown). Control staining of tonsils revealed pDCs evenly dis-persed in the T cell areas (Fig. 8C). In situ analysis of chemokinereceptor expression revealed homogeneous CXCR3 expression inpDCs as well as in the majority of the cells infiltrating HSV-in-duced lesions (Fig. 8D; pDCs are yellow). Other chemokine re-ceptors (e.g., CCR5, CCR7, CXCR4) were either not detectable orwere identified on a minor part of the cellular infiltrate only (datanot shown).

HSV and IFN-� regulate CXCR3 and CCR7 surface expressionby pDCs

To investigate the effects of HSV exposure on the migratory ca-pacity of pDCs, we cultured these cells in the presence of HSV anddetermined chemokine receptor expression and function. Within6 h of HSV exposure, pDCs started to lose CXCR3 and to gainCCR7 expression (Fig. 8E). After 12 h, the loss of CXCR3 wascomplete and the expression of CCR7 was at its peak (Fig. 8, E andG). Consistent with the rapid and massive gain of CCR7 expres-sion, pDCs started to migrate to soluble gradients of CCL19 within6 h of virus exposure (Fig. 8F). Exposure of pDCs to exogenousIFN-� and to influenza virus down-regulated CXCR3 and inducedCCR7 expression and responsiveness to a similar magnitude asseen with HSV (Fig. 8H, data not shown). Thus, the direct contactwith different viruses and/or virus-induced nIFNs stimulates theloss of CXCR3 and up-regulation of CCR7-dependent migration.In in situ analyses of HSV-induced lesions, pDCs were occasion-ally found in close proximity to podoplanin-expressing lymphaticvessels (Fig. 8I). This indicates that HSV-induced CCR7 expres-sion allows pDCs to exit the tissue and to migrate toward regionallymph nodes via CCR7 ligand-expressing lymphatic vessels (25).

DiscussionA successful combat of viral infection requires at least two com-ponents: the secretion of virus replication-inhibiting factors suchas nIFNs and the induction of virus-specific T cell immunity. pDCsare ideally suited to accomplish both these tasks. To understandtheir functionality in vivo it is important to learn whether and howpDCs enter virus-infected tissues. This study provides original ev-idence that CXCR3L immobilization on ECs may be required andsufficient to target pDCs into foci of viral replication.

It has been known that CXCR3 is a predominant chemokinereceptor expressed on circulating pDCs (Refs. 6, 12, 14; thisstudy). In functional terms, however, CXCR3 is inactive in induc-ing chemotaxis of pDCs (Refs. 12, 14, and 19; this study). Recentwork emphasizes that CXCR3 functions only as a threshold reg-ulator of CXCR4-driven chemotaxis (14). In this study, however,we show that the CXCR3-CXCR3L interaction is fully competentto induce adhesion and migration of pDCs. These events areevoked only when CXCR3Ls are presented to pDCs in solid phase,a physiologically relevant condition. Glycosaminoglycans, HS, inparticular, are crucial for productive presentation of chemokines invivo (29, 31, 32). Our study shows that CXCR3Ls which are pre-sented by HS on artificial surfaces or the plasma membrane ofECs, but again not the soluble chemokines, can deliver the promi-gratory signals to pDCs.

Intriguing is the observation that migration in response to im-mobilized CXCR3Ls is a cell type-restricted phenomenon.CXCR3� T cells, in contrast to pDCs, failed to migrate toCXCR3Ls immobilized on artificial membranes and apical EC sur-faces but migrated vigorously to soluble chemokine gradients. Thisimplies that CXCR3 acquires the specific functionality to respond

FIGURE 5. HS-dependent CXCL10 binding to ECsand EC-associated CXCR3 ligands in HSV-infected skin.A, CXCL10 binds to HS moieties on ECs and competeswith CXCL12 for EC binding. ECs were either pretreated(middle panels) or not pretreated with heparitinase (toppanels, bottom panel) and pulsed with CXCL10 orCXCL12 (100 ng/ml each). Chemokine binding was re-vealed by sequential incubations with anti-CXCL10 oranti-CXCL12 Abs and Alexa 488-conjugated second stepreagents. In the bottom panel, ECs were pretreated withCXCL12 before CXCL10 pulsing and anti-CXCL10 im-munostaining. Ab reactivity was measured by FACS (x-axis). Mean fluorescence intensities (MFI) were calcu-lated by subtracting the MFI of nonpulsed ECs from theMFI of chemokine-pulsed ECs (top and middle panels) orby subtracting the MFI of CXCL12 preincubated andCXCL10-pulsed ECs from the MFI of CXCL10-pulsedECs (bottom panel). B, Dose dependency of CXCL10binding to ECs. ECs were incubated with the indicatedconcentrations of CXCL10 and binding was analyzed byFACS. Data are given as mean MFI values (�SD) asobtained in four independent experiments. �, Significant(p � 0.05) CXCL10 binding. C, CXCR3 ligands areexpressed in HSV-infected skin. Immunohistochemicalstaining for CXCL9 (upper left panel), CXCL10 (lowerleft panel), and CXCL11 (upper right panel) of 5-�mcryosections from gluteal herpes lesions reveals expres-sion of all three CXCR3 ligands in the epidermal layers.Moreover, CXCL10 and CXCL11 but not CXCL9 immu-noreactivity was found associated with ECs of the dermalvascular plexus (insets in the respective panels). Isotypecontrol mAb staining of HSV lesions (lower right panel)and anti-CXCR3 ligand stainings of unaffected skin (datanot shown) revealed no immunoreactivity.

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to immobilized chemokines only in the context of the pDCmembrane microenvironment. Mechanistically, transmigration ofpDCs takes place only to apically immobilized chemokine, signi-fying the involvement of a negative step gradient. Positive hapto-tactic CXCR3L gradients as well as chemokines immobilized in anongradient fashion failed to induce the transmigration of pDCs.

Thus, the postulated mechanism of pDC migration includes twomechanistic facets: the migration to immobilized chemokine (hap-totaxis) and the movement along a negative chemokine gradient(chemorepulsion). These two migratory mechanisms have been de-scribed for other chemokines and other leukocyte types (30, 38,39). However, until now, in sharp contrast to the pDC response toCXCR3Ls, the chemokine-induced haptotaxis and chemorepulsionwere shown to take place separately from each other and only asalternatives to also viable chemotaxis. Remarkably, in the case of

CXCR3L-induced pDC migration, “haptorepulsion” is the onlypossible in vitro mechanism of the observed response. Analogouschemokine immobilization-dependent migration has been de-scribed in T cells in response to CXCL12 which, however, alsorequires lateral shear stress (40). Lateral shear stress is not requiredfor TEM of pDCs. Conceivably, haptorepulsion, as shown forpDCs as a paradigmatic example in this study, may be a novelrelevant migratory mechanism used by other cell types in responseto different chemokines.

CXCL12 is clearly capable of being productively presented byECs to pDCs in a similar fashion as CXCL10. Thus, our datasuggest that CXCR4, besides being a receptor mediating chemo-taxis, may be able to execute haptorepulsion in pDCs. Our dataalso show that CXCR3 and CXCR4 on pDCs can function

FIGURE 6. pDCs but not T cells transmigrate through CXCL10-pre-senting EC monolayers. Monolayers of ECs were grown on collagen gels.ECs were cultured in the presence (activated ECs, �) or the absence (rest-ing ECs, f) of 100 U/ml TNF-� for 4 h. EC monolayers were washed andpulsed or not pulsed with CXCL10 or CXCL12 (100 ng/ml). A and B,pDCs adhere to and transmigrate across chemokine-pulsed, non-activatedEC monolayers. Cell Tracker Green-labeled pDCs were layered on top ofthe modified or nonmodified ECs and allowed to adhere to (A) and totransmigrate through the EC monolayers (B) for 3 h. Mean baseline adhe-sion and migration of pDCs correspond to 80 and 11 cells/HPF, respec-tively. �, p � 0.01; ��, p � 0.05 as compared with respective mediumcontrols. C, Granulocytes transmigrate through activated but not throughnon-activated EC monolayers. Cell Tracker Green-labeled PMN cells wereseeded onto and allowed to transmigrate across the EC monolayers for 30min. Mean baseline migration of granulocytes across resting ECs corre-sponds to 1 cell/HPF. D, Poor transmigration of T cells through CXCL10-presenting EC monolayers. Cell Tracker Green-labeled IL-2-activated Tcells were layered on top of the modified or nonmodified ECs and allowedto transmigrate through the monolayers for 3 h. Adhesion (A) and TEM (B,C, and D) of pDCs (A and B), PMN cells (C), and T cells (D) were mea-sured by assessing the numbers of Cell Tracker Green-labeled cells (andCD123-coexpressing cells in the case of pDCs) on the apical EC surfaceand within the subendothelial collagen matrix by multilevel confocal LSMand automated cell counting. Results are depicted as mean increase inmigration/adhesion (�SD) relative to the migration/adhesion induced byresting, non-chemokine-modified ECs (x-axis).

FIGURE 7. CXCL10 presentation-dependent, Gi-protein-mediatedTEM of pDCs is prevented by soluble chemokines. A, Inhibition ofCXCL10-mediated EC adhesion and TEM of pDCs by soluble CXCR3ligands. Nonmodified pDCs or pDCs prepulsed with CXCL10 (1–100 ng/ml), CXCL9, or CXCL11 (100 ng/ml each) were allowed to adhere to andto transmigrate across CXCL10-loaded EC monolayers. Result are ex-pressed as the mean percent inhibition (�SD) of TEM (f) and adhesion(�). Zero percent (0%) inhibition corresponds to the migration/adhesion ofnonpulsed pDCs on CXCL10-loaded ECs. B, Heterologous desensitizationof chemokine presentation-dependent EC adhesion and TEM. Nonmodi-fied pDCs or pDCs prepulsed with CXCL10 or CXCL12 (100 ng/ml each)were allowed to adhere to and to transmigrate across CXCL10- orCXCL12-loaded EC monolayers. Result are expressed as the mean percentinhibition (�SD) of TEM (f) and adhesion (�). Zero percent (0%) inhi-bition corresponds to the migration/adhesion of nonpulsed pDCs onCXCL10- or CXCL12-loaded ECs. Note that soluble CXCL12 abolishesCXCL10-dependent responses and that CXCL10 as well as CXCL12 pre-vent CXCL12-dependent adhesion and TEM. C and D, CXCL10 presen-tation-dependent TEM is Gi protein-mediated while CXCL10 presentation-dependent EC adhesion of pDCs is a Gi protein-independent event. pDCswere exposed (f) or were not exposed (�) to PTX before layering themonto CXCL10-loaded or non-loaded EC monolayers. Depicted are the ad-hesion (C) and migration indices (D) of pDCs relative to the adhesion/migration of non-PTX-treated pDCs on non-chemokine-pulsed ECs.

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independently from each other. This argument derives from ex-periments showing that CXCL10 bound to artificial membranessuffices to deliver the migratory signal, that primary ECs that sup-port pDC migration via immobilized CXCL10 lack CXCL12 im-munoreactivity, and that blocking anti-CXCR4 mAbs do not in-terfere with CXCL10-mediated migration across ECs (data notshown). Our data, however, do not exclude the possibility that the

two chemokine receptors use a common signal transduction machin-ery to execute pDC haptorepulsion. In support of the latter stands theobservation of heterologous desensitization of CXCR3- and CXCR4-dependent pDC responses by the respective ligands. However, in thescenario of a viral infection, the possible importance of the CXCR4/CXCL12-dependent migration will be diminished due to the appear-ance of virally induced CXCR3Ls that compete with CXCL12 for EC

FIGURE 8. pDCs accumulate in HSV-induced skin lesions. A and B, Perivascular localization of pDCs in HSV lesions. Cryostat sections obtained fromHSV-2-infected gluteal skin were subjected to CD123-Oregon Green (green), CD4-PE (red), and CD45RA-Cy5 (blue; confocal LSM overlay image in A)or to CD123-Oregon Green (green), CD4-PE (red), and CD8-Cy5 (blue) triple immunolabeling (overlay image in B). pDCs, i.e., CD123�CD4�CD45RA�

cells (whitish cells in A) and CD123�CD4�CD8� cells (yellow cells in B), are assembled in small clusters juxtaposed to CD123� dermal blood vessels(BV, green structures in A and B). CD4� (red cells in A and B) and CD8� T cells (blue cells in B) are loosely arranged around the pDC clusters. C, Stainingof tonsillar tissue with CD4 (red), CD123 (green), and CD45RA (blue)-specific mAbs. pDCs do not occur in clusters but are equally distributed in the Tcell-rich areas. D, Predominance of CXCR3� cells in the cellular infiltrate of HSV-induced skin lesions. Virtually all CD123� pDCs express CXCR3(yellow cells). CD123� cells in the inflammatory infiltrate also express CXCR3 (red cells). E, Rapid reciprocal regulation of CXCR3 and CCR7 expressionon pDCs by HSV exposure. Purified pDCs were exposed to HSV-2 (1 � 106 PFU/ml) for the indicated time periods (x-axis) and analyzed for CXCR3 andCCR7 expression by FACS (MFI values, y-axis). F, HSV-induced up-regulation of CCR7 coincides with the rapid induction of pDC chemotaxis to solublegradients of CCL19. pDCs were cultured for the indicated time period in the presence of HSV-2 and tested for their ability to chemotactically respond toCCL19 or CXCL10 (100 ng/ml each). Migration indices (y-axis) are shown as a function of time of HSV exposure (x-axis). G, HSV is a potent stimulantof pDC maturation. pDCs were cultured in the presence of IL-3/TNF-� (upper panel) or HSV-2 (lower panel) for 12 h and analyzed for the expressionof the maturation marker CCR7 and CD83 (closed histograms; left and right panel, respectively). HSV-2 promoted pDC maturation even more potentlythan the classical maturation mixture IL-3 plus TNF-�. Dashed lines indicate cut-off levels for positive immunofluorescence as revealed by isotype-matchedcontrol mAb staining (open histograms). H, HSV, influenza virus, and exogenous IFN-� are equally potent inducers of CCR7-dependent chemotacticactivity in pDCs. Freshly isolated pDCs (n.s.), or pDCs exposed to IL-3 plus TNF-�, IFN-�, HSV-2, or influenza A virus/PR8 strain were tested for theirability to chemotactically respond to CCL19 or CXCL10 in transwell insert chemotaxis assays. Migration indices are given on the y-axis. I, Juxtapositionof pDCs and lymphatic vessels in HSV lesions. Sections were stained with lymphatic vessel (LV)-specific anti-podoplanin Abs (red). pDCs were identifiedby coexpression of CD45RA (green) and CD123 (blue).

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binding (Fig. 5, A and C). The involvement of CXCR3 in pDCmigration into sites of viral replication is further substantiated bythe finding that pDCs express the “classical” form of CXCR3 andnot its migration-incompetent B variant (26) as revealed by RT-PCR, cDNA cloning, and sequence analysis.

Our contention that CXCR3L-binding and presenting moietiesexist on ECs derives from the observations that CXCR3Ls arepresent on ECs in cutaneous sites of viral replication and thatCXCR3Ls bind primary ECs in a dose-dependent and saturablefashion. At the first glance, candidate EC receptors includeCXCR3B that binds CXCR3Ls with lower affinity than CXCR3A(26). The observations, however, that CXCR3L binding to ECs isHS-dependent and that low concentrations of CXCL12 can inhibitEC CXCR10 binding speak in favor of a previously not recognizedCXCR10L-binding structure on ECs.

pDCs more efficiently adhered to and migrated across restingthan activated EC monolayers. Shedding of the HS-based chemo-kine binding sites during EC activation could be responsible forthis phenomenon (41) but the actual mechanism remains to bedetermined. More importantly, our data strongly suggest that ac-tivation-induced up-regulation of EC adhesion molecules is notrequired for TEM of pDCs. Granulocytes, in contrast to pDCs,migrated across activated ECs selectively. We speculate that theabundant expression of L-selectin, selectin ligands including CLAand PSGL-1 as well as ligands for intestinal addressins (Refs. 6, 8,and 14; this study) may allow pDCs to roll on resting or on min-imally activated ECs and, thus, to constantly screen peripheralsites and lymph nodes for chemokine signatures of viral infection.In this respect, the CXCR3Ls may be of particular significance asthey are among the gene products that are extremely rapidly androbustly up-regulated after viral encounter (42, 43). In aggregate,these observations allow for speculation that pDC traffic into vi-rally infected sites is maximal at early time points and may de-crease with the onset of overt EC activation and EC activation-dependent recruitment of effector cells of the innate and adaptiveimmune system.

Soluble chemokines exert a negative signal to pDCs that is dom-inant over the productive proadhesive and migratory signal in-duced by immobilized chemokines. This suggests a novel thera-peutic possibility to prevent pDC homing to peripheral tissues inautoimmune diseases such as lupus erythematosus and function-ally related pathologies such as graft rejection (44–46). To thisend, CXC chemokines genetically engineered to preserve theirCXCR3-desensitizing capacity but to abolish their EC/HS-bindingability may be used to prevent pDC-mediated tissue damage.

Upon HSV exposure, pDCs almost instantaneously down-regu-late CXCR3 and up-regulate CCR7 and respond to its ligands che-motactically. Our previous results demonstrated that CCR7Ls aresecreted basolaterally by lymphatic ECs (25) and, thus, may be inthe position to guide the virus-exposed pDCs into the lymphaticchannels. In summary, the two distinctive mechanisms of pDCtrafficking, the CXCR3L immobilization-dependent, haptorepul-sive migration and the chemotactic gradient-induced departure de-fine consecutive stages of maturation-related pDC functionality.

AcknowledgmentsWe thank B. Reininger and E. Schwarzinger for excellent technicalassistance.

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