of October 19, 2015.This information is current as

TLR2Human Dendritic Cells via Triggering of

Production inγMycobacteria Induce IFN-

and Sven BrandauLehan, Holger Heine, Torsten Goldmann, Andreas Böhle Ingo Fricke, Daniell Mitchell, Jessica Mittelstädt, Nadine

http://www.jimmunol.org/content/176/9/5173doi: 10.4049/jimmunol.176.9.5173

2006; 176:5173-5182; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/176/9/5173.full#ref-list-1

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Mycobacteria Induce IFN-� Production in Human DendriticCells via Triggering of TLR21

Ingo Fricke,2,3* Daniell Mitchell,2* Jessica Mittelstadt,* Nadine Lehan,* Holger Heine,†

Torsten Goldmann,‡ Andreas Bohle,§ and Sven Brandau4*

IFN-� is of central importance for the induction of robust cell-mediated immunity and for the activation of APC. Recent studiesusing experimental murine systems have now suggested a fundamental role for APC-derived IFN-� during infection with intra-cellular pathogens. It is currently unknown whether human dendritic cells (DC) can respond to bacterial stimulation with pro-duction of IFN-�. To test this question, we used human monocyte-derived DC stimulated by Mycobacterium bovis bacillusCalmette-Guerin as a model system. We demonstrate production of IFN-� mRNA and protein on the single cell level. IFN-� inDC cultures was not simply produced by contaminating lymphocytes because production of DC-IFN-� could also be demonstratedin highly purified DC cultures containing virtually no T, B, and NK cells. TLR2 was identified as a key receptor involved intriggering production of DC-IFN-�. Interestingly, DC-IFN-� seems to participate in an autocrine DC activation loop, and pro-duction of DC-IFN-� could be enhanced by costimulation of DC with IL-12/IL-15/IL-18. In conclusion, we have demonstratedproduction of IFN-� by human DC on the single cell level, identified TLR2 as a pattern recognition receptor involved in thisprocess, and elucidated some of the functional consequences of autocrine IFN-� production by human DC. The Journal ofImmunology, 2006, 176: 5173–5182.

D endritic cells (DC)5 patrol peripheral tissues as sentinelsof the immune system and they are of crucial importancefor the induction of cellular immunity against intracel-

lular pathogens including mycobacteria (1). Several lines of evi-dence have demonstrated that DC are the major APC for initiationof primary T cell responses as well as the initial source of IL-12 inmicrobial infections (2–4). Mycobacterium tuberculosis and M.bovis bacillus Calmette-Guerin (BCG) infection of human or mu-rine myeloid DC induces a coordinate process of cell maturationand up-regulation of IL-12 production (5, 6). Subsequent transferof BCG-infected DC into mice led to rapid IFN-� responsesagainst mycobacterial Ags (5), and M. tuberculosis-infected DCinduced potent immunity against experimental tuberculosis inmice (7). The Th1 cytokine IFN-� has been identified as a keycytokine controlling mycobacterial infections and is produced byboth CD4 and CD8 T cells (8, 9) in infected individuals as well asby NK cells (10, 11). To date, IFN-� knockout (GKO) mice areamong the most susceptible to challenge with virulent M. tuber-

culosis (12, 13), and individuals defective in genes for IFN-� orthe IFN-�R are prone to serious mycobacterial infections (14).Altogether, these data underscore the crucial and essential role forIFN-� in antimycobacterial immunity.

Although T cells and NK cells have long been regarded as theexclusive source for IFN-� in mycobacterial infections, recent re-ports suggest that IFN-� might also be produced by monocytes/macrophages and professional APC (15–17). In these studies IL-12and/or microbial stimulation have been identified as the most po-tent inducers of IFN-� production by macrophages and DC (17,18). Consequently, accumulating evidence on production of IFN-�by APC has led other investigators to postulate the so-called “jumpstart” model of APC activation (19). Although in earlier studiesproduction of IFN-� by APC has largely been demonstrated invitro using cultured macrophages and DC (19), recent studies us-ing intact intracellular pathogens (20) or synthetic �-galactosylce-ramide (21) have now provided additional evidence for the pro-duction of IFN-� under in vivo conditions in murine modelsystems.

A caveat and common criticism to some approaches has beenthe possible contribution of contaminating lymphocytic cells toIFN-� production in myeloid cell cultures (22) and the measure-ment of IFN-� mRNA but not IFN-� protein in stimulated APC(23). Also most studies report on the use of murine macrophagesand until now it is completely unclear whether human DC alsohave the capacity to produce IFN-�. To fill some of the study gapswe aim to analyze production of both IFN-� mRNA and protein byhuman DC on the single cell level, to specifically address the roleof contaminating lymphocytes, and to test the relevance of theproposed jump start model for human professional APC.

In the present report we demonstrate production of IFN-�mRNA and protein by human DC after mycobacterial challenge onthe single cell level. Furthermore, we identified TLR2 as a patternrecognition receptor (PRR) involved in this process and elucidatedsome of the functional consequences of autocrine IFN-� produc-tion by human DC. Thus, our data support a role for professional

*Division of Immunotherapy, †Division of Innate Immunity, and ‡Division of Pa-thology, Research Center Borstel, Borstel, Germany; and §Helios Agnes Karll Hos-pital, Bad Schwartau, Germany

Received for publication May 18, 2005. Accepted for publication February 6, 2006.

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 in part by Deutsche Forschungsgemeinschaft,Sonderforschungsbereich 367, Project C7, and Graduiertenkolleg 288, Project B9 andProject C8.2 I.F. and D.M. contributed equally to this work.3 Current address: H. Lee Moffitt Cancer Center, University of South Florida, MoffittResearch Center Building Room 2067, 12902 Magnolia Drive, Tampa, FL 33612.4 Address correspondence and reprint requests to Dr. Sven Brandau, Division of Im-munotherapy, Research Center Borstel, Parkallee 1-40, 23845 Borstel, Germany. E-mail address: sbrandau@fz-borstel.de5 Abbreviations used in this paper: DC, dendritic cell; BCG, bacillus Calmette-Guerin; PRR, pattern recognition receptor; MOI, multiplicity of infection; HEK, hu-man embryonic kidney.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

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APC as early sources for IFN-� after microbial stimulation and areconsistent with an autocrine DC activation model upon bacterialencounter.

Materials and MethodsAbs, cytokines, and reagents

The mAb directed against CD1a-PE was acquired from Immunotech andanti-CD209 was obtained from eBioscience (BioCarta). Anti-CD14-PE andanti-CD86-PE mAbs were purchased from Dianova. Anti-CD83 and anti-mouse MHC class II were obtained from BD Pharmingen and IgG1-PE andgoat anti-mouse-PE were obtained from DakoCytomation. Anti-IFN-�(GZ-4) mAb was bought from Bender MedSystems. Abs against TLR2(TLR2.31.2) were a gift from Genentech (proposal no. 203745). BlockingmAb against IFN-�R1 as well as azide-free IgG1 and IgG2a isotype con-trols were purchased from BD Pharmingen. Synthetic TLR4 antagonisttetra acyl lipid A (compound 406) and LPS (Salmonella minnesota, com-pound 201) were a gift from Prof. K. Brandenburg (Research CenterBorstel, Borstel, Germany). Recombinant human GM-CSF, IL-4, IL-12,and IL-15 were purchased from PeproTech-Tebu. IL-18 was bought fromCell Sciences. Human serum was obtained from AB� donors from theInstitute of Immunology and Transfusion Medicine (University of Lu-ebeck, Luebeck, Germany). RPMI 1640 medium (Invitrogen Life Tech-nologies) supplemented with 10% heat-inactivated FCS (Linaris), L-glu-tamine (2 mM), penicillin (100 U/ml), and streptomycin (100 �g/ml) wasused as the R.10 complete culture medium. BCG (strain Connaught) usedfor stimulation of DC was from the logarithmic growth phase of cultures inliquid Middlebrook 7H9.

DC generation and purification

Human DC. Human PBMC from heparinized blood of healthy donorswere prepared by Ficoll-Paque (Biochrom) density gradient centrifugation.Monocytes and lymphocytes were then separated by counter flow elutria-tion from PBMC. Afterward, purity of resulting monocytes and lympho-cytes was determined by light scatter analysis to be �95% in all cases.Immature human DC were generated according to Sallusto and Lanzavec-chia (24). In brief, monocytes were cultured for 7 days in R.10 completemedium supplemented with GM-CSF (500 U/ml) and IL-4 (500 U/ml),exchanging half of the medium including cytokines every 2–3 days. Tofurther purify DC generated from elutriated monocytes, contaminatinglymphocytes were depleted using a commercially available system of mag-netic bead-coupled specific Abs (MACS; Miltenyi Biotec). Briefly, on day7 of the differentiation process, DC were mixed with magnetic bead-cou-pled anti-CD3, anti-CD19, and anti-CD56 mAbs and applied to an LScolumn (Miltenyi Biotec). Depletion of T cells, B cells, and NK cells wasconfirmed by flow cytometry.Murine DC. Bone-marrow cells were isolated from wild-type control andTLR2-deficient mice (25) (provided by Amgen, and Institute of MedicalMicrobiology, Immunology and Hygiene, Technical University, Munich,Germany). Bone marrow-derived DC were generated essentially as de-scribed by Lutz et al. (26) and stimulated with BCG at a multiplicity ofinfection (MOI) � 10.

Flow cytometric analysis

APC were stained with PE-conjugated mAbs in PBS-3% human serum,washed, fixed with 1.5% paraformaldehyde and analyzed on a FACSCali-bur using the CellQuest software (BD Biosciences). Data analysis wasdone using WinMDI 2.8 (by J. Trotter, The Scripps Research Institute, LaJolla, CA).

Cytokine ELISA

The concentration of TNF-� in culture supernatants was determined witha quantitative ELISA, provided by Dr. H. Gallati (Intex, Muttenz, Swit-zerland) and performed as recommended by the manufacturer. IFN-� in theculture supernatants was determined by OptEIA Human ELISA kits (BDPharmingen) according to the manufacturer’s instructions. IL-12 was de-termined with the eBioscience Human IL-12p70 ELISA Ready-SET-Go!kit from BioCarta.

Cytokine secretion assay

For isolation of IFN-� secreting DC, DC were stimulated with BCG(MOI � 1) for 12 h and applied to an IFN-� secretion assay (cell enrich-ment and detection kit; Miltenyi Biotec) as described by the manufacturer,and using a 1-h cytokine secretion period. In principle in this assay, se-creted IFN-� protein is immobilized at the cell membrane of the secretory

cell with a bispecific catch reagent reacting with CD45 and IFN-�. Leu-kocytes secreting IFN-� are then isolated and enriched with a combinationof magnetic bead-coupled Abs directed against the cell-associated IFN-�.

Cytokine production by DC

DC were stimulated with BCG (MOI � 1) or LPS (10 ng/ml) for 24 h inthe presence of anti-TLR2 mAb (10 �g/ml), IgG2a (10 �g/ml), or com-pound 406 (1 �g/ml) added 30 min before stimulation. To elucidate theeffects of cytokines on BCG-mediated IFN-� production, DC were stim-ulated with a suboptimal dose of BCG in the presence of IL-12, IL-15, orIL-18 or combinations thereof. To determine the effects of IFN-� on DCactivation, inhibitory mAbs to IFN-�R1 (10 �g/ml) or IgG1 (10 �g/ml)were added 30 min before BCG treatment. Other MOI values besidesMOI � 1 were also tested for stimulation of DC. MOI � 1 has been chosenas a compromise between stronger activation but impaired viability athigher MOI values.

Activation of transiently transfected human embryonic kidney(HEK) cells

HEK293 cells were plated at a density of 5 � 104/ml in 96-well plates incomplete DMEM without G418. The following day, cells were transientlytransfected using Polyfect (Qiagen) according to the manufacturer’s pro-tocol. Expression plasmid containing human CD14 was a gift from Dr.D. T. Golenbock (University of Massachusetts Medical School, Worcester,MA), and the Flag-tagged versions of human TLR2 and human TLR4 werea gift from P. Nelson (University of Washington, Seattle, WA) and sub-cloned into pREP9 (Invitrogen Life Technologies). The human MD-2 ex-pression plasmid was a gift from K. Miyake (University of Tokyo, Tokyo,Japan). Plasmids were used at 200 ng (25 ng for CD14 and MD-2) pertransfection. The total DNA content was kept constant at 450 ng per trans-fection using pCDNA3 (Invitrogen Life Technologies). After 24 h, HEK-TLR2-CD14 and HEK-MD-2-TLR4-CD14 were washed and stimulatedwith BCG or TLR ligands for another 18 h. Finally, supernatants werecollected and IL-8 content was quantified by ELISA (BioSourceInternational).

In situ hybridization

In situ hybridization was performed as described by Umland et al. (27). Inbrief, HOPE (DCS Innovative Diagnostik Systeme) fixed cytospins wereincubated in acetone-glyoxal followed by dehydration in acetone and iso-propanol and rehydration of air-dried slides with DEPC water (diethylpyrocarbonate). Overnight hybridization was performed with freshly de-naturated digoxigenated DNA probe, yeast tRNA (Roche), 0.1% SDS, and50% formamide in PBS. The hybrids were detected after stringency wash-ing by an alkaline phosphatase-conjugated anti-digoxigenin Ab using NewFuchsin as a chromogen. Cells were counterstained with hematoxylin.

Confocal fluorescence microscopy

Labeling of surface CD209 Ag (rat anti-CD209; eBioscience) on cytospinpreparations was followed by fixation and permeabilization and finally byintracellular staining for IFN-� (mouse anti-IFN-�, clone GZ4; BenderMedSystems). For fluorescence tagging, goat anti-rat Alexa Fluor 488(Molecular Probes) and goat anti-mouse Alexa Fluor 546 (MolecularProbes) coupled Abs were used. Nuclear staining was obtained by TOTO-3(Molecular Probes). BCG was labeled with Syto45 (Molecular Probes)before stimulation according to manufacturer’s instructions. Optical sec-tions were analyzed with a Leica TCS SP2 confocal microscope.

ResultsActivation of DC by BCG mycobacteria

In a first series of experiments we assessed some general pheno-typic and functional changes of human monocyte-derived DC afterstimulation with BCG mycobacteria. As observed by others (6, 28)mycobacterial stimulation led to up-regulation of costimulatorymolecules (CD86) and maturation markers (CD83, MHC class II)on DC concomitant with down-regulation of CD209, which hasbeen identified as a receptor for BCG mycobacteria on DC (29, 30)(Fig. 1). This phenotypic maturation was also accompanied byfunctional maturation as BCG-stimulated DC showed a strongerinduction of lymphoproliferation in MLR, induction of lympho-cytic cytokines, and induction of cytolytic NK activity when com-pared with immature unstimulated DC (31 and data not shown).

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Production of IFN-� by crude and purified DC

In a first attempt to investigate production of IFN-� by human DC,we detected production of IFN-� mRNA in cultures of BCG-stim-ulated DC (data not shown). As such signal could well be the resultof amplification of mRNA from contaminating lymphocytes, wenext searched for the presence of T cells, B cells, and NK cells aspossible sources for IFN-� in our DC cultures. Using monocytecultures of 95–99% purity for DC generation we detected small butclearly detectable subpopulations of lymphocytes in DC culturesafter 7 days of differentiation (termed crude DC in Fig. 2A). Con-taminating lymphocytes were mostly CD3-positive T cells,whereas only very small numbers of contaminating CD56-positiveNK cells were present. As we wanted to investigate cytokine pro-

duction of DC in the absence of even minute amounts of lympho-cytes, we designed a purification protocol for crude DC cultures.Positive isolation of DC with magnetic beads coupled to DC-spe-cific Ags like MHC class II, CD1a, or CD209 was not feasible,resulted in low purity, and compromised integrity and viability ofDC cultures (data not shown). In contrast, negative isolation of DCby magnetic depletion of contaminating lymphocytes resulted inDC cultures of high purity and viability and these cultures were es-sentially free of contaminating T cells, B cells, and NK cells (termedpurified DC in Fig. 2A). Therefore, populations of purified DC gen-erated by this procedure were used in subsequent experiments.

Because initially we had observed production of IFN-� mRNAin cultures of crude DC, we next investigated production of IFN-�

FIGURE 1. Activation and maturation of DC afterchallenge with Mycobacterium bovis BCG. Peripheralblood monocytes were cultured for 7 days in the pres-ence of GM-CSF and IL-4 (each 500 U/ml) followed by3 days stimulation with BCG (MOI � 1). Top row, Un-stimulated DC; bottom row, DC stimulated with BCG.Isotype controls (gray filled histogram) and staining inten-sity of respective specific mAbs (open histogram) areshown. Mean fluorescence intensity (as difference betweenisotype and specific Ab) is indicated. One representativeexperiment of six with comparable results is shown.

FIGURE 2. Production of IFN-�by DC after challenge with BCG. A,Monocyte-derived DC were analyzedby flow cytometry for the presence ofcontaminating lymphocytes with (pu-rified) or without (crude) additionalmagnetic depletion of CD3-positive,CD19-positive, and CD56-positivecells. Note the absence of contami-nating lymphocytes in the indicatedregion of purified DC compared withisotype controls and with crude DC.B, Crude and purified DC were pre-pared as in A and left unstimulated(iDC) or were stimulated with BCG(DC�BCG) for 24 h. Secretion ofIFN-� was measured by ELISA. Onerepresentative experiment of three isshown.

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protein in crude and purified DC cultures stimulated with BCGmycobacteria. Although we measured induction of IFN-� proteinin crude DC cultures we also reproducibly detected IFN-� proteinin cultures of stimulated purified DC (mostly in a range from 50 to500 pg/ml) (Fig. 2B). In most experiments IFN-� secretion in cul-tures of crude DC exceeded production of IFN-� in purified DCcultures. These data suggested to us that production of IFN-� incultures of BCG-stimulated DC could not merely be the result ofcontaminating lymphocytic cells, although lymphocytes seem toconsiderably contribute to the secretion of IFN-� into the super-natant of crude DC cultures.

To ascertain IFN-� production by human DC we next wanted todemonstrate production of this cytokine by DC on the single celllevel. For this purpose we performed cytokine secretion assays, insitu hybridization and confocal microscopy. We subjected un-stimulated and BCG-stimulated DC to a magnetic bead-based cy-tokine secretion assay. In this assay IFN-� is retained at the cellmembrane of the secretory cell and IFN-�-positive cells can sub-sequently be enriched on affinity columns by means of magneticbeads. Flow-through and eluate cells were analyzed. As expected,flow-through cells did not express IFN-� (data not shown). IFN-�positive cells were eluated when BCG-stimulated DC were appliedto the column, whereas in contrast unstimulated DC remainedIFN-� negative (Fig. 3A). Although this experiment demonstratedIFN-� production by DC on the cell population level we proceededin our studies with in situ hybridization and confocal microscopyto correlate IFN-� signal with single cell images of DC. In situhybridization revealed clear signals for IFN-� mRNA in BCG-stimulated DC, whereas unstimulated DC and control hybridiza-tions in stimulated DC remained negative (Fig. 3B). To demon-strate that IFN-� mRNA is also translated into protein, cytospinswere subjected to immunocytofluorescence for IFN-� protein. Asshown in Fig. 3C, BCG-stimulated DC stained positive for IFN-�,whereas unstimulated DC and isotype control stainings were neg-ative. Identity of IFN-�-positive DC was further confirmed by dou-ble-immunofluorescence for IFN-� and CD209 (DC-SIGN), a sur-face Ag, which is specifically expressed on certain subsets ofhuman DC, including monocyte-derived DC. Continuously, up-take of BCG by IFN-� positive DC was observed. By analyzing alarge number of confocal images combined with flow cytometricanalyses of DC infected with BCG expressing GFP (32) we de-termined that the majority of DC (�90%) expressed IFN-�,whereas the rate of BCG-positive infected DC was in a range from20 to 50% in these experiments (Fig. 3D). Thus using single cellassays, our experiments for the first time provide clear evidence forthe production of the important Th1 cytokine IFN-� by human DC.

Role of TLR2 in DC activation and IFN-� production

TLR2 and TLR4 have been suggested as receptors for mycobac-teria on human APC (33–35). Thus, we examined 1) whether BCG

preparations used in our study are recognized by either TLR2 orTLR4 and 2) the role of the respective TLRs in BCG-mediatedactivation of DC and induction of DC-derived IFN-�.

By means of reporter gene assays using TLR-transfected HEKcells we could clearly demonstrate TLR2-dependent cellular acti-vation by BCG. BCG preparations induced IL-8 secretion in HEK-TLR2 transfectants at least to the same extent as Pam3CysSK4, asynthetic lipopeptide and well-defined ligand for TLR2. In con-trast, BCG preparations did not induce cytokine secretion in HEK-TLR4 transfectants, suggesting a preferential recognition of BCGby TLR2 vs TLR4 (Fig. 4A). Involvement of TLR2 in BCG-me-diated DC activation was then confirmed using bone marrow-de-rived DC from wild-type and TLR2-deficient mice. In these ex-periments production of TNF-� as well as up-regulation of MHCclass II were severely impaired in BCG-stimulated DC fromTLR2-deficient mice compared with DC from wild-type mice (Fig.4B). To examine whether TLR2 is also involved in BCG-mediatedactivation of human DC we stimulated human DC with BCG in thepresence of inhibitory Abs to TLR2 and measured the release ofTNF-� and IL-12p70. As shown in Fig. 4C the induction of bothcytokines was significantly inhibited in the presence of anti-TLR2Abs. Inhibition by anti-TLR2 was specific as isotype control Abshad no effect on DC activation and anti-TLR2 Abs had no effect onLPS-induced TLR2-independent activation of DC. Inhibition ofTLR4 signaling by compound 406 did not alter cytokine responsesof BCG-stimulated DC. Next we tested whether triggering ofTLR2 would also be required for induction of DC-IFN-�. In BCG-stimulated cultures we observed a significant inhibition of DC-derived IFN-� after addition of anti-TLR2 Abs, whereas LPS-in-duced IFN-� remained unchanged (Fig. 4D). In contrast, inhibitionof TLR4 signaling by compound 406 did not reduce BGG-inducedproduction of DC-IFN-�. We conclude from these experimentsthat TLR2 is of crucial importance for BCG-mediated activation ofhuman DC and for induction of DC-IFN-� production. Interest-ingly, LPS, which is described as a very potent inducer of DCmaturation, activation, and cytokine release, seems to be only aweak inducer of DC-derived IFN-�. Thus, in the majority of ourexperiments BCG-induced IFN-� exceeds LPS-induced IFN-�whereas LPS is a much more potent inducer of TNF-� and other“classical” DC cytokines (Fig. 4, C and D).

Autocrine DC activation and regulation of DC IFN-�

So far we have demonstrated production of IFN-� by human DCon the single cell level and we have elucidated the role of the PRRTLR2 in this process. In the final part of this study we wanted toexamine the regulation of DC-IFN-� and a possible role of thiscytokine in autocrine DC activation based on a recently proposedso-called jump start model (19).

To investigate whether DC-IFN-� regulates its own expression,we stimulated DC with BCG in the presence of inhibitory Abs to

FIGURE 3. IFN-� production by DC can be detected on the single cell level. A, BCG-stimulated (MOI � 1) and unstimulated DC were analyzed bycytokine secretion assay. Flow cytometric detection of IFN-� is shown for cells magnetically retained on the column (eluate). Flow-through cells did notexpress IFN-� (data not shown). B, Cytospin preparations of unstimulated (iDC) or 6-h BCG-stimulated DC (MOI � 1) (DC�BCG) were prepared. In situhybridization with an IFN-� probe or irrelevant vector probe is shown. Nuclear staining was done with hematoxylin, and detection of hybridization productwas done by New Fuchsin. C, Confocal microscopy images of either BCG-stimulated or unstimulated DC (MOI � 1, 18 h). Staining for DC-SIGN/CD209is shown in green (Alexa Fluor 488), and staining for IFN-� appears in red (Alexa Fluor 546). Staining of the nucleus (TOTO-3) and BCG (Syto45) appearsin blue. BCG staining was performed before DC stimulation. Separate pictures of nuclear staining and BCG staining were added in two different colorchannels, and the merged image was transferred into the blue channel of the image. Merging of images and adjustment of contrast and brightness were donewith Adobe Photoshop. Images were taken on a Leica TCS SP2 confocal microscope. D, DC were stimulated with GFP-transfected BCG (MOI � 1), andbinding/uptake of bacteria was quantified (left) by FACS analysis after 4 h of coculture. BCG binding was observed in a range from 20 to 50% of totalDC (interexperimental variation). In parallel, cytospin preparations of DC stimulated with untransfected BCG (MOI � 1) were prepared, immunostained(as for C), and analyzed by confocal microscopy (right). Note that the majority of DC show IFN-� signals, whereas in only 5 of 12 cells ingested BCGbacteria can be visualized. One representative experiment of three (A and B), of six (C), and of four (D) is shown.

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IFN-�R1. However, secretion of DC-IFN-� remained essentiallyunchanged after blockade of the IFN-�R indicating that productionof DC-IFN-� is not under autoregulatory control (Fig. 5A). Wethen wanted to know whether DC-IFN-� would be involved in theregulation of other cytokines released by DC after mycobacterialstimulation. As shown in Fig. 5B this was indeed the case as in-hibition of the IFN-�R1 during BCG stimulation resulted in re-duced production of TNF-�. Consequently, when we added exog-enous recombinant IFN-� to BCG-stimulated DC, production ofTNF-� as well as of IL-12 could be further induced (Fig. 5C).IL-12 in combination with IL-18 and/or IL-15 has been describedto modulate IFN-� production in murine DC, lymphocytes, andmacrophages. In our study these cytokines further induced BCG-mediated production of DC-IFN-�. Although single cytokines onlymoderately enhanced BCG-induced production of DC-IFN-�,IFN-� secretion was significantly up-regulated by additional stim-ulation with cytokine combinations (IL-12/IL-15 and IL-12/IL-18)(Fig. 6A). Stimulation of DC with cytokines alone for 24 h in theabsence of BCG did not induce IFN-� production (Fig. 6A). How-ever, low amounts of DC-IFN-� could be measured after pro-longed (up to 3 days) stimulation of DC with cytokines alone (datanot shown). Importantly, further up-regulation of BCG-inducedDC-IFN-� by cytokines could not only be observed in culture su-

pernatants of stimulated DC, but was also detectable on the singlecell level by confocal microscopy (Fig. 6B).

Taken together the data of the final part of our study indicatethat DC-IFN-� is part of a cytokine network secreted by humanDC encountering mycobacterial pathogens. Within this networkDC-IFN-� plays a role in autocrine DC activation as it regulatesthe production of other cytokines, namely TNF-�.

DiscussionIFN-� is a central mediator of cellular Th1 immunity and abso-lutely required for effective immunity against intracellular patho-gens including mycobacteria. Thus, IFN-� gene disrupted mice areunable to control growth of M. tuberculosis and succumb to oth-erwise sublethal infectious doses of mycobacteria (12, 13). In ad-dition, IFN-� also plays an important role in the activation of APCleading to both activation of antibacterial cellular responses andenhanced induction of antibacterial lymphocyte responses (36).Consequently, the question whether also APC themselves couldproduce IFN-� gained considerable interest (19).

After early reports on IFN-� production by human alveolar mac-rophages (15, 37) subsequent studies mostly focused on murineAPC. Experiments with in vitro generated or ex vivo isolated mu-rine APC demonstrated production of significant amounts of

FIGURE 4. TLR2 is involved in BCG-mediated DCactivation. A, TLR2 and TLR4 interaction with wholeBCG bacteria was analyzed using the respective HEKtransfectants. HEK cells expressing human TLR2 orTLR4 were stimulated with BCG (lyoBCG; Immucyst),synthetic lipopeptide (Pam3CysSK4, defined TLR2 li-gand), LPS (TLR2-independent defined TLR4 ligand),and TNF-� (TLR-independent). IL-8 secretion wasquantitated by ELISA and IL-8 produced by controlHEK cells lacking TLR was subtracted as background.B, Bone marrow-derived DC from wild-type control(WT) and TLR2-deficient (KO) mice were stimulatedwith BCG. Secretion of TNF-� and MHC class II meanfluorescence intensity (MFI) expression by unstimulatedand BCG-stimulated DC was measured. C and D, Inhi-bition of DC cytokine secretion by inhibition of TLR2.Purified human DC were stimulated with BCG or LPSand 30 min before stimulation anti-TLR2 mAb (Ab; 10�g/ml), isotype control mAb (Iso; 10 �g/ml), or syn-thetic TLR4 antagonist tetra acyl lipid A precursor lipidIVa compound 406 (406; 1 �g/ml) were added. Super-natants were taken 24 h past stimulation and analyzedfor TNF-� (left) and IL-12p70 (right) and IFN-� (D).Statistical difference was calculated using Student’s ttest. One representative experiment of three (A and B)and of six (C and D) with comparable results is shown.

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IFN-� by these cells (38, 39). Recently, there has also been in-creasing evidence for the production of IFN-� by myeloid cells invivo based on experimental murine systems (20, 21). In a largenumber of these studies cytokines (mostly IL-12 and IL-18) andintracellular pathogens have been identified as potent inducers ofDC-derived IFN-�. Based on these findings, a so-called jump startmodel for APC activation has been proposed (19).

Despite growing evidence for IFN-� production by murine my-eloid cells until now virtually nothing is known about the potencyof human APC to produce this important cytokine. Also, produc-tion of IFN-� by APC is still a matter of intensive debate becausea significant number of studies did not report on production ofIFN-� protein but rather focused on production of mRNA only(23). Moreover, most investigators reported on macrophages ratherthan DC and the contribution of contaminating lymphocytes has

not always been clarified explicitly (22). Thus, this is the first studyto provide a detailed analysis on the expression of IFN-� by humanDC on the single cell level, to address the role of contaminatinglymphocytes in this regard, and to elucidate some of the functionalconsequences of autocrine DC-IFN-� production. Using a cytokinesecretion assay, in situ hybridization, and confocal microscopy wewere able to detect mRNA and protein expression of IFN-� inhuman DC. In addition to pure morphology, identity of DC wasconfirmed by immunocytochemistry and the IFN-�-producingcells coexpressed DC-SIGN, a surface molecule specifically ex-pressed on monocyte-derived DC and not present on lymphocytesor NK cells. Depending on the protocol for in vitro differentiationof monocyte-derived DC, starting cultures of monocytes can havea purity ranging from well below 80% up to almost 99% (40–42).In most of our experiments for standard DC differentiation, weobtained a DC purity between 90 and 97% with CD3-positive Tcells being the most frequent contaminating cell population (seeFig. 2A, crude DC). Using a magnetic bead based Ab depletionsystem we succeeded in generating DC populations of high purityvirtually devoid of contaminating lymphocytes and NK cells (seeFig. 2A, purified DC). Comparison of IFN-� production in culturesof crude and purified DC revealed higher cytokine levels in cul-tures of crude DC. Nevertheless, we consistently observed signif-icant levels of IFN-� protein also in cultures of purified DC. To-gether with the single cell analysis by confocal microscopy thesedata clearly indicate that human DC have the capacity to produceIFN-� protein. At the same time these data also show that con-taminating lymphocytes most likely contribute to the production ofIFN-� in most cultures of conventionally generated monocyte-de-rived human DC. Parallel detection of IFN-� expression and BCGinfection revealed a considerable proportion of DC, which wereIFN-�-positive and BCG-negative. At first glance these findingssuggest that direct infection is not absolutely essential for cytokineproduction. However, we have to consider that BCG uptake pre-cedes cytokine production by many hours and mycobacteria mightalready be degraded in a proportion of IFN-�-positive DC.

After we had demonstrated production of IFN-� by human DCstimulated with BCG mycobacteria, we next wanted to elucidatesome of the mechanistic and functional implications of this find-ing. TLR represents a family of ancient sensing molecules ex-pressed on a variety of cell types and belong to a group of recep-tors commonly referred to as PRR (43). TLR2 has been identifiedas a major TLR mediating recognition of mycobacteria by immunecells. However, the relative contribution of different TLRs (namelyTLR2 and TLR4) to antimycobacterial immunity under in vivoconditions remains controversial and might well depend on themycobacterial strain used, the dose, and route of infection and thetiming of the experiment (34, 44–46). Nevertheless, in vitro stim-ulation of murine myeloid cells with whole intact BCG mycobac-teria seems to be preferentially mediated by TLR2 (33, 34, 44).Interestingly, extracts consisting of BCG cell wall skeleton andpeptidoglycan extracted from BCG bacteria are dependent on bothTLR2 and TLR4 signaling for activation of myeloid cells (47, 48).In our hands, TLR2 was clearly dominant over TLR4 in conferringcellular activation in response to BCG mycobacteria. HEK cellsoverexpressing TLR2 were strongly activated by BCG and thisactivation was fully comparable to the response achieved by thewell-defined synthetic lipopeptide PAM3CysSK4. In contrast, noTLR4-dependent activation could be observed in HEK cells over-expressing TLR4. Similarly, blocking of TLR2 on human DCclearly reduced their cytokine production, and most important pro-duction of DC-IFN-�, in response to BCG. At the same time,

FIGURE 5. Autocrine IFN-� and BCG cooperate in the induction ofcytokines. DC were generated from peripheral blood monocytes, purifiedby magnetic depletion of lymphocytes, and stimulated with BCG or leftuntreated. At 30 min before BCG treatment either a blocking Ab to IFN-�R1 (�IFN-�R1) or an isotype control (IgG1) were added (10 �g/ml).IFN-� (A) and TNF-� (B) in the culture supernatant were determined byELISA. C, To examine the effect of exogenous IFN-� on BCG-inducedcytokine release, DC were stimulated with a suboptimal dose of BCG(MOI � 0.5) or left unstimulated (iDC) and increasing doses of recombi-nant human IFN-� were added. TNF-� and IL-12 production were deter-mined by ELISA. One representative experiment of six with comparableresults is shown.

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blockade of TLR4 signaling did not substantially alter the immu-nological response of BCG-stimulated human DC. Given the com-plexity of the interaction of potential pathogen-associated molec-ular patterns on the surface of BCG mycobacteria with theirreceptors on human DC, it was not surprising that in subsequentexperiments production of DC-IFN-� was not completely blockedin the presence of inhibitory Abs to TLR2. We would also like tonote that stimulation of DC with the synthetic TLR2 ligandPAM3CysSK4 induced only low amounts of cytokines in DC ex-cept for IL-6 (data not shown). This finding is consistent withprevious reports in which additional costimulation with IFN-� wasrequired for full DC activation and IL-12 production (49). Com-bined, these data suggest that TLR2 is of crucial importance for theinduction of DC-IFN-� by BCG mycobacteria, but additional re-ceptors might also participate in this process. In addition, theseexperiments suggested to us the interesting possibility that DC-IFN-� could function as an autocrine amplifier and costimulator ofTLR-mediated responses in human DC.

Thus, in the final part of our study we have investigated a pos-sible autocrine amplification loop mediated by DC-IFN-�. Indeed,our experiments are consistent with such a process as inhibition ofthe IFN-�R resulted in reduced production of TNF-� after BCG

stimulation. Interestingly, IFN-� does not regulate its own expres-sion as blocking of IFN-�R did not change BCG-induced levels ofDC-IFN-�. In the context of a possible autocrine IFN-�-drivenAPC activation model it is of note that for M. tuberculosis it hasbeen described that the 19-kDa lipoprotein inhibits IFN-�-depen-dent activation of murine macrophages (50, 51) if prolonged TLR2ligation precedes IFN-� signaling. It remains to be defined whethersuch effects are also effective in human professional APC such asDC or whether they are limited to macrophages, which are pri-marily involved in immediate host defense and pathogen elimina-tion. Alternatively, these data offer the interesting possibility thatvirulent mycobacterial strains have developed mechanisms to cir-cumvent autocrine APC activation by DC-IFN-� as a possible wayto down-regulate or inhibit full immune activation.

A difference between murine and human APC was noted withregard to the effects of IFN-�-inducing cytokines. Although in mu-rine macrophages and DC IL-12 alone or in combination withIL-18 has been described as a potent inducer of DC-derived IFN-�(18, 52), in our study in the absence of mycobacterial challenge,these cytokines induced human DC-IFN-� only after prolongedperiods of stimulation (3 days). Nonetheless, IL-12, IL-15, and

FIGURE 6. IFN-� production byDC can be synergistically enhancedby a combined stimulation with BCGand IL-12/IL-15/IL-18. Purified DCwere stimulated with a suboptimaldose of BCG (MOI � 0.5) or left un-treated. IL-12, IL-15, or IL-18 (10ng/ml each) were added in combina-tion or alone as indicated. A, Super-natants were harvested 24 h paststimulation, and IFN-� content wasdetermined by ELISA. B, Usingsome cultures of stimulated DC, cy-tospin preparations were prepared,immunostained (as for Fig. 3C) andanalyzed by confocal microscopy.Note the enhanced signal for IFN-�in DC costimulated with BCG andcytokines. Isotype control stainingswere negative and similar to Fig. 3C.One representative experiment ofthree with comparable results isshown.

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IL-18 and combinations thereof, were profound costimulators forinduction of DC-IFN-� when combined with BCG stimulation.

In our study we have demonstrated for the first time productionof the “lymphocytic” cytokine IFN-� by human DC. Recently,there has been accumulating evidence for the production of an-other lymphocytic cytokine, namely IL-2, by APC. Although ini-tial studies focused on murine APC (53), IL-2 production has nowalso been observed in human DC. Interestingly, in human myeloid,DC production of IL-2 strongly depends on costimulation withIL-15 (54). Thus, our study supports growing evidence that certaincytokines like IL-2 and IFN-�, which have previously beenthought to be exclusively produced by lymphocytes, might also besecreted by APC. In this regard we have no doubt that lymphocyteswill produce higher amounts of IFN-� per cell compared with DC.Nonetheless production of even small amounts of lymphocytic cy-tokines by APC could still have a significant impact on immuneresponses in the respective micromilieu.

In conclusion, in this study we have described DC-derivedIFN-� as a cytokine participating in a complex autocrine activationloop after mycobacterial stimulation. These findings might be ofimportant relevance for the mechanistic comprehension of theearly phases of immune responses against intracellular pathogenslike mycobacteria.

AcknowledgmentsWe especially thank Gabriele Bentien for brilliant technical assistancethroughout the entire project. We thank Dr. Oliver Umland for the prepa-ration of the IFN-� probe used for in situ hybridization, Erika Kaltenhauserand Renate Bergmann for elutriation of monocytes and TNF-� ELISA, andHeike Kuhl and Prof. E. Vollmer for support with in situ hybridization. Weare grateful to Genentech for providing the anti-TLR2 Ab, we acknowledgethe help of U. Wolfram (Miltenyi Biotec) with the cytokine secretion assay,and are grateful to Dr. Thomas Scholzen for skillful introduction into con-focal microscopy techniques. Finally, we thank Prof. Stefan Ehlers forcritical reading and helpful comments on the manuscript.

DisclosuresThe authors have no financial conflict of interest.

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