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The Journal of Immunology, 2006, 176: 5173-5182.
Copyright © 2006 by The American Association of Immunologists

Mycobacteria Induce IFN-{gamma} Production in Human Dendritic Cells via Triggering of TLR21

Ingo Fricke2,3,*, Daniell Mitchell2,*, Jessica Mittelstädt*, Nadine Lehan*, Holger Heine{dagger}, Torsten Goldmann{ddagger}, Andreas Böhle§ and Sven Brandau4,*

* Division of Immunotherapy, {dagger} Division of Innate Immunity, and {ddagger} Division of Pathology, Research Center Borstel, Borstel, Germany; and § Helios Agnes Karll Hospital, Bad Schwartau, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IFN-{gamma} is of central importance for the induction of robust cell-mediated immunity and for the activation of APC. Recent studies using experimental murine systems have now suggested a fundamental role for APC-derived IFN-{gamma} during infection with intracellular pathogens. It is currently unknown whether human dendritic cells (DC) can respond to bacterial stimulation with production of IFN-{gamma}. To test this question, we used human monocyte-derived DC stimulated by Mycobacterium bovis bacillus Calmette-Guérin as a model system. We demonstrate production of IFN-{gamma} mRNA and protein on the single cell level. IFN-{gamma} in DC cultures was not simply produced by contaminating lymphocytes because production of DC-IFN-{gamma} could also be demonstrated in highly purified DC cultures containing virtually no T, B, and NK cells. TLR2 was identified as a key receptor involved in triggering production of DC-IFN-{gamma}. Interestingly, DC-IFN-{gamma} seems to participate in an autocrine DC activation loop, and production of DC-IFN-{gamma} could be enhanced by costimulation of DC with IL-12/IL-15/IL-18. In conclusion, we have demonstrated production of IFN-{gamma} by human DC on the single cell level, identified TLR2 as a pattern recognition receptor involved in this process, and elucidated some of the functional consequences of autocrine IFN-{gamma} production by human DC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)5 patrol peripheral tissues as sentinels of the immune system and they are of crucial importance for the induction of cellular immunity against intracellular pathogens including mycobacteria (1). Several lines of evidence have demonstrated that DC are the major APC for initiation of primary T cell responses as well as the initial source of IL-12 in microbial infections (2, 3, 4). Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) infection of human or murine myeloid DC induces a coordinate process of cell maturation and up-regulation of IL-12 production (5, 6). Subsequent transfer of BCG-infected DC into mice led to rapid IFN-{gamma} responses against mycobacterial Ags (5), and M. tuberculosis-infected DC induced potent immunity against experimental tuberculosis in mice (7). The Th1 cytokine IFN-{gamma} has been identified as a key cytokine controlling mycobacterial infections and is produced by both CD4 and CD8 T cells (8, 9) in infected individuals as well as by NK cells (10, 11). To date, IFN-{gamma} knockout (GKO) mice are among the most susceptible to challenge with virulent M. tuberculosis (12, 13), and individuals defective in genes for IFN-{gamma} or the IFN-{gamma}R are prone to serious mycobacterial infections (14). Altogether, these data underscore the crucial and essential role for IFN-{gamma} in antimycobacterial immunity.

Although T cells and NK cells have long been regarded as the exclusive source for IFN-{gamma} in mycobacterial infections, recent reports suggest that IFN-{gamma} might also be produced by monocytes/macrophages and professional APC (15, 16, 17). In these studies IL-12 and/or microbial stimulation have been identified as the most potent inducers of IFN-{gamma} production by macrophages and DC (17, 18). Consequently, accumulating evidence on production of IFN-{gamma} by APC has led other investigators to postulate the so-called "jump start" model of APC activation (19). Although in earlier studies production of IFN-{gamma} by APC has largely been demonstrated in vitro using cultured macrophages and DC (19), recent studies using intact intracellular pathogens (20) or synthetic {alpha}-galactosylceramide (21) have now provided additional evidence for the production of IFN-{gamma} under in vivo conditions in murine model systems.

A caveat and common criticism to some approaches has been the possible contribution of contaminating lymphocytic cells to IFN-{gamma} production in myeloid cell cultures (22) and the measurement of IFN-{gamma} mRNA but not IFN-{gamma} protein in stimulated APC (23). Also most studies report on the use of murine macrophages and until now it is completely unclear whether human DC also have the capacity to produce IFN-{gamma}. To fill some of the study gaps we aim to analyze production of both IFN-{gamma} mRNA and protein by human DC on the single cell level, to specifically address the role of contaminating lymphocytes, and to test the relevance of the proposed jump start model for human professional APC.

In the present report we demonstrate production of IFN-{gamma} mRNA and protein by human DC after mycobacterial challenge on the single cell level. Furthermore, we identified TLR2 as a pattern recognition receptor (PRR) involved in this process and elucidated some of the functional consequences of autocrine IFN-{gamma} production by human DC. Thus, our data support a role for professional APC as early sources for IFN-{gamma} after microbial stimulation and are consistent with an autocrine DC activation model upon bacterial encounter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs, cytokines, and reagents

The mAb directed against CD1a-PE was acquired from Immunotech and anti-CD209 was obtained from eBioscience (BioCarta). Anti-CD14-PE and anti-CD86-PE mAbs were purchased from Dianova. Anti-CD83 and anti-mouse MHC class II were obtained from BD Pharmingen and IgG1-PE and goat anti-mouse-PE were obtained from DakoCytomation. Anti-IFN-{gamma} (GZ-4) mAb was bought from Bender MedSystems. Abs against TLR2 (TLR2.31.2) were a gift from Genentech (proposal no. 203745). Blocking mAb against IFN-{gamma}R1 as well as azide-free IgG1 and IgG2a isotype controls were purchased from BD Pharmingen. Synthetic TLR4 antagonist tetra acyl lipid A (compound 406) and LPS (Salmonella minnesota, compound 201) were a gift from Prof. K. Brandenburg (Research Center Borstel, Borstel, Germany). Recombinant human GM-CSF, IL-4, IL-12, and IL-15 were purchased from PeproTech-Tebu. IL-18 was bought from Cell Sciences. Human serum was obtained from AB+ donors from the Institute of Immunology and Transfusion Medicine (University of Luebeck, Luebeck, Germany). RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (Linaris), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) was used as the R.10 complete culture medium. BCG (strain Connaught) used for stimulation of DC was from the logarithmic growth phase of cultures in liquid Middlebrook 7H9.

DC generation and purification

Human DC. Human PBMC from heparinized blood of healthy donors were prepared by Ficoll-Paque (Biochrom) density gradient centrifugation. Monocytes and lymphocytes were then separated by counter flow elutriation from PBMC. Afterward, purity of resulting monocytes and lymphocytes was determined by light scatter analysis to be ≥95% in all cases. Immature human DC were generated according to Sallusto and Lanzavecchia (24). In brief, monocytes were cultured for 7 days in R.10 complete medium supplemented with GM-CSF (500 U/ml) and IL-4 (500 U/ml), exchanging half of the medium including cytokines every 2–3 days. To further purify DC generated from elutriated monocytes, contaminating lymphocytes were depleted using a commercially available system of magnetic bead-coupled specific Abs (MACS; Miltenyi Biotec). Briefly, on day 7 of the differentiation process, DC were mixed with magnetic bead-coupled anti-CD3, anti-CD19, and anti-CD56 mAbs and applied to an LS column (Miltenyi Biotec). Depletion of T cells, B cells, and NK cells was confirmed by flow cytometry.

Murine DC. Bone-marrow cells were isolated from wild-type control and TLR2-deficient mice (25) (provided by Amgen, and Institute of Medical Microbiology, Immunology and Hygiene, Technical University, Munich, Germany). Bone marrow-derived DC were generated essentially as described by Lutz et al. (26) and stimulated with BCG at a multiplicity of infection (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 FACSCalibur using the CellQuest software (BD Biosciences). Data analysis was done using WinMDI 2.8 (by J. Trotter, The Scripps Research Institute, La Jolla, CA).

Cytokine ELISA

The concentration of TNF-{alpha} in culture supernatants was determined with a quantitative ELISA, provided by Dr. H. Gallati (Intex, Muttenz, Switzerland) and performed as recommended by the manufacturer. IFN-{gamma} in the culture supernatants was determined by OptEIA Human ELISA kits (BD Pharmingen) according to the manufacturer’s instructions. IL-12 was determined with the eBioscience Human IL-12p70 ELISA Ready-SET-Go! kit from BioCarta.

Cytokine secretion assay

For isolation of IFN-{gamma} secreting DC, DC were stimulated with BCG (MOI = 1) for 12 h and applied to an IFN-{gamma} secretion assay (cell enrichment and detection kit; Miltenyi Biotec) as described by the manufacturer, and using a 1-h cytokine secretion period. In principle in this assay, secreted IFN-{gamma} protein is immobilized at the cell membrane of the secretory cell with a bispecific catch reagent reacting with CD45 and IFN-{gamma}. Leukocytes secreting IFN-{gamma} are then isolated and enriched with a combination of magnetic bead-coupled Abs directed against the cell-associated IFN-{gamma}.

Cytokine production by DC

DC were stimulated with BCG (MOI = 1) or LPS (10 ng/ml) for 24 h in the presence of anti-TLR2 mAb (10 µg/ml), IgG2a (10 µg/ml), or compound 406 (1 µg/ml) added 30 min before stimulation. To elucidate the effects of cytokines on BCG-mediated IFN-{gamma} production, DC were stimulated with a suboptimal dose of BCG in the presence of IL-12, IL-15, or IL-18 or combinations thereof. To determine the effects of IFN-{gamma} on DC activation, inhibitory mAbs to IFN-{gamma}R1 (10 µg/ml) or IgG1 (10 µg/ml) were added 30 min before BCG treatment. Other MOI values besides MOI = 1 were also tested for stimulation of DC. MOI = 1 has been chosen as a compromise between stronger activation but impaired viability at higher MOI values.

Activation of transiently transfected human embryonic kidney (HEK) cells

HEK293 cells were plated at a density of 5 x 104/ml in 96-well plates in complete DMEM without G418. The following day, cells were transiently transfected using Polyfect (Qiagen) according to the manufacturer’s protocol. 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 were a gift from P. Nelson (University of Washington, Seattle, WA) and subcloned into pREP9 (Invitrogen Life Technologies). The human MD-2 expression 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) per transfection. The total DNA content was kept constant at 450 ng per transfection using pCDNA3 (Invitrogen Life Technologies). After 24 h, HEK-TLR2-CD14 and HEK-MD-2-TLR4-CD14 were washed and stimulated with BCG or TLR ligands for another 18 h. Finally, supernatants were collected and IL-8 content was quantified by ELISA (BioSource International).

In situ hybridization

In situ hybridization was performed as described by Umland et al. (27). In brief, HOPE (DCS Innovative Diagnostik Systeme) fixed cytospins were incubated in acetone-glyoxal followed by dehydration in acetone and isopropanol and rehydration of air-dried slides with DEPC water (diethyl pyrocarbonate). Overnight hybridization was performed with freshly denaturated digoxigenated DNA probe, yeast tRNA (Roche), 0.1% SDS, and 50% formamide in PBS. The hybrids were detected after stringency washing by an alkaline phosphatase-conjugated anti-digoxigenin Ab using New Fuchsin as a chromogen. Cells were counterstained with hematoxylin.

Confocal fluorescence microscopy

Labeling of surface CD209 Ag (rat anti-CD209; eBioscience) on cytospin preparations was followed by fixation and permeabilization and finally by intracellular staining for IFN-{gamma} (mouse anti-IFN-{gamma}, clone GZ4; Bender MedSystems). For fluorescence tagging, goat anti-rat Alexa Fluor 488 (Molecular Probes) and goat anti-mouse Alexa Fluor 546 (Molecular Probes) 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 sections were analyzed with a Leica TCS SP2 confocal microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Activation of DC by BCG mycobacteria

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


Figure 1
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  		FIGURE 1. Activation and maturation of DC after challenge with Mycobacterium bovis BCG. Peripheral blood monocytes were cultured for 7 days in the presence of GM-CSF and IL-4 (each 500 U/ml) followed by 3 days stimulation with BCG (MOI = 1). Top row, Unstimulated DC; bottom row, DC stimulated with BCG. Isotype controls (gray filled histogram) and staining intensity of respective specific mAbs (open histogram) are shown. Mean fluorescence intensity (as difference between isotype and specific Ab) is indicated. One representative experiment of six with comparable results is shown.

 
Production of IFN-{gamma} by crude and purified DC

In a first attempt to investigate production of IFN-{gamma} by human DC, we detected production of IFN-{gamma} mRNA in cultures of BCG-stimulated DC (data not shown). As such signal could well be the result of amplification of mRNA from contaminating lymphocytes, we next searched for the presence of T cells, B cells, and NK cells as possible sources for IFN-{gamma} in our DC cultures. Using monocyte cultures of 95–99% purity for DC generation we detected small but clearly detectable subpopulations of lymphocytes in DC cultures after 7 days of differentiation (termed crude DC in Fig. 2A). Contaminating lymphocytes were mostly CD3-positive T cells, whereas only very small numbers of contaminating CD56-positive NK cells were present. As we wanted to investigate cytokine production of DC in the absence of even minute amounts of lymphocytes, we designed a purification protocol for crude DC cultures. Positive isolation of DC with magnetic beads coupled to DC-specific Ags like MHC class II, CD1a, or CD209 was not feasible, resulted in low purity, and compromised integrity and viability of DC cultures (data not shown). In contrast, negative isolation of DC by magnetic depletion of contaminating lymphocytes resulted in DC cultures of high purity and viability and these cultures were essentially free of contaminating T cells, B cells, and NK cells (termed purified DC in Fig. 2A). Therefore, populations of purified DC generated by this procedure were used in subsequent experiments.


Figure 2
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  		FIGURE 2. Production of IFN-{gamma} by DC after challenge with BCG. A, Monocyte-derived DC were analyzed by flow cytometry for the presence of contaminating lymphocytes with (purified) or without (crude) additional magnetic depletion of CD3-positive, CD19-positive, and CD56-positive cells. Note the absence of contaminating lymphocytes in the indicated region of purified DC compared with isotype controls and with crude DC. B, Crude and purified DC were prepared as in A and left unstimulated (iDC) or were stimulated with BCG (DC+BCG) for 24 h. Secretion of IFN-{gamma} was measured by ELISA. One representative experiment of three is shown.

 
Because initially we had observed production of IFN-{gamma} mRNA in cultures of crude DC, we next investigated production of IFN-{gamma} protein in crude and purified DC cultures stimulated with BCG mycobacteria. Although we measured induction of IFN-{gamma} protein in crude DC cultures we also reproducibly detected IFN-{gamma} protein in cultures of stimulated purified DC (mostly in a range from 50 to 500 pg/ml) (Fig. 2B). In most experiments IFN-{gamma} secretion in cultures of crude DC exceeded production of IFN-{gamma} in purified DC cultures. These data suggested to us that production of IFN-{gamma} in cultures of BCG-stimulated DC could not merely be the result of contaminating lymphocytic cells, although lymphocytes seem to considerably contribute to the secretion of IFN-{gamma} into the supernatant of crude DC cultures.

To ascertain IFN-{gamma} production by human DC we next wanted to demonstrate production of this cytokine by DC on the single cell level. For this purpose we performed cytokine secretion assays, in situ hybridization and confocal microscopy. We subjected unstimulated and BCG-stimulated DC to a magnetic bead-based cytokine secretion assay. In this assay IFN-{gamma} is retained at the cell membrane of the secretory cell and IFN-{gamma}-positive cells can subsequently be enriched on affinity columns by means of magnetic beads. Flow-through and eluate cells were analyzed. As expected, flow-through cells did not express IFN-{gamma} (data not shown). IFN-{gamma} positive cells were eluated when BCG-stimulated DC were applied to the column, whereas in contrast unstimulated DC remained IFN-{gamma} negative (Fig. 3A). Although this experiment demonstrated IFN-{gamma} production by DC on the cell population level we proceeded in our studies with in situ hybridization and confocal microscopy to correlate IFN-{gamma} signal with single cell images of DC. In situ hybridization revealed clear signals for IFN-{gamma} mRNA in BCG-stimulated DC, whereas unstimulated DC and control hybridizations in stimulated DC remained negative (Fig. 3B). To demonstrate that IFN-{gamma} mRNA is also translated into protein, cytospins were subjected to immunocytofluorescence for IFN-{gamma} protein. As shown in Fig. 3C, BCG-stimulated DC stained positive for IFN-{gamma}, whereas unstimulated DC and isotype control stainings were negative. Identity of IFN-{gamma}-positive DC was further confirmed by double-immunofluorescence for IFN-{gamma} and CD209 (DC-SIGN), a surface Ag, which is specifically expressed on certain subsets of human DC, including monocyte-derived DC. Continuously, uptake of BCG by IFN-{gamma} positive DC was observed. By analyzing a large number of confocal images combined with flow cytometric analyses of DC infected with BCG expressing GFP (32) we determined that the majority of DC (>90%) expressed IFN-{gamma}, whereas the rate of BCG-positive infected DC was in a range from 20 to 50% in these experiments (Fig. 3D). Thus using single cell assays, our experiments for the first time provide clear evidence for the production of the important Th1 cytokine IFN-{gamma} by human DC.


Figure 3
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  		FIGURE 3. IFN-{gamma} production by DC can be detected on the single cell level. A, BCG-stimulated (MOI = 1) and unstimulated DC were analyzed by cytokine secretion assay. Flow cytometric detection of IFN-{gamma} is shown for cells magnetically retained on the column (eluate). Flow-through cells did not express IFN-{gamma} (data not shown). B, Cytospin preparations of unstimulated (iDC) or 6-h BCG-stimulated DC (MOI = 1) (DC+BCG) were prepared. In situ hybridization with an IFN-{gamma} probe or irrelevant vector probe is shown. Nuclear staining was done with hematoxylin, and detection of hybridization product was done by New Fuchsin. C, Confocal microscopy images of either BCG-stimulated or unstimulated DC (MOI = 1, 18 h). Staining for DC-SIGN/CD209 is shown in green (Alexa Fluor 488), and staining for IFN-{gamma} appears in red (Alexa Fluor 546). Staining of the nucleus (TOTO-3) and BCG (Syto45) appears in blue. BCG staining was performed before DC stimulation. Separate pictures of nuclear staining and BCG staining were added in two different color channels, and the merged image was transferred into the blue channel of the image. Merging of images and adjustment of contrast and brightness were done with Adobe Photoshop. Images were taken on a Leica TCS SP2 confocal microscope. D, DC were stimulated with GFP-transfected BCG (MOI = 1), and binding/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 total DC (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-{gamma} signals, whereas in only 5 of 12 cells ingested BCG bacteria can be visualized. One representative experiment of three (A and B), of six (C), and of four (D) is shown.

 
Role of TLR2 in DC activation and IFN-{gamma} production

TLR2 and TLR4 have been suggested as receptors for mycobacteria on human APC (33, 34, 35). Thus, we examined 1) whether BCG preparations used in our study are recognized by either TLR2 or TLR4 and 2) the role of the respective TLRs in BCG-mediated activation of DC and induction of DC-derived IFN-{gamma}.

By means of reporter gene assays using TLR-transfected HEK cells we could clearly demonstrate TLR2-dependent cellular activation by BCG. BCG preparations induced IL-8 secretion in HEK-TLR2 transfectants at least to the same extent as Pam3CysSK4, a synthetic lipopeptide and well-defined ligand for TLR2. In contrast, BCG preparations did not induce cytokine secretion in HEK-TLR4 transfectants, suggesting a preferential recognition of BCG by TLR2 vs TLR4 (Fig. 4A). Involvement of TLR2 in BCG-mediated DC activation was then confirmed using bone marrow-derived DC from wild-type and TLR2-deficient mice. In these experiments production of TNF-{alpha} as well as up-regulation of MHC class II were severely impaired in BCG-stimulated DC from TLR2-deficient mice compared with DC from wild-type mice (Fig. 4B). To examine whether TLR2 is also involved in BCG-mediated activation of human DC we stimulated human DC with BCG in the presence of inhibitory Abs to TLR2 and measured the release of TNF-{alpha} and IL-12p70. As shown in Fig. 4C the induction of both cytokines was significantly inhibited in the presence of anti-TLR2 Abs. Inhibition by anti-TLR2 was specific as isotype control Abs had no effect on DC activation and anti-TLR2 Abs had no effect on LPS-induced TLR2-independent activation of DC. Inhibition of TLR4 signaling by compound 406 did not alter cytokine responses of BCG-stimulated DC. Next we tested whether triggering of TLR2 would also be required for induction of DC-IFN-{gamma}. In BCG-stimulated cultures we observed a significant inhibition of DC-derived IFN-{gamma} after addition of anti-TLR2 Abs, whereas LPS-induced IFN-{gamma} remained unchanged (Fig. 4D). In contrast, inhibition of TLR4 signaling by compound 406 did not reduce BGG-induced production of DC-IFN-{gamma}. We conclude from these experiments that TLR2 is of crucial importance for BCG-mediated activation of human DC and for induction of DC-IFN-{gamma} production. Interestingly, LPS, which is described as a very potent inducer of DC maturation, activation, and cytokine release, seems to be only a weak inducer of DC-derived IFN-{gamma}. Thus, in the majority of our experiments BCG-induced IFN-{gamma} exceeds LPS-induced IFN-{gamma} whereas LPS is a much more potent inducer of TNF-{alpha} and other "classical" DC cytokines (Fig. 4, C and D).


Figure 4
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  		FIGURE 4. TLR2 is involved in BCG-mediated DC activation. A, TLR2 and TLR4 interaction with whole BCG bacteria was analyzed using the respective HEK transfectants. HEK cells expressing human TLR2 or TLR4 were stimulated with BCG (lyoBCG; Immucyst), synthetic lipopeptide (Pam3CysSK4, defined TLR2 ligand), LPS (TLR2-independent defined TLR4 ligand), and TNF-{alpha} (TLR-independent). IL-8 secretion was quantitated by ELISA and IL-8 produced by control HEK cells lacking TLR was subtracted as background. B, Bone marrow-derived DC from wild-type control (WT) and TLR2-deficient (KO) mice were stimulated with BCG. Secretion of TNF-{alpha} and MHC class II mean fluorescence intensity (MFI) expression by unstimulated and BCG-stimulated DC was measured. C and D, Inhibition of DC cytokine secretion by inhibition of TLR2. Purified human DC were stimulated with BCG or LPS and 30 min before stimulation anti-TLR2 mAb (Ab; 10 µg/ml), isotype control mAb (Iso; 10 µg/ml), or synthetic TLR4 antagonist tetra acyl lipid A precursor lipid IVa compound 406 (406; 1 µg/ml) were added. Supernatants were taken 24 h past stimulation and analyzed for TNF-{alpha} (left) and IL-12p70 (right) and IFN-{gamma} (D). Statistical difference was calculated using Student’s t test. One representative experiment of three (A and B) and of six (C and D) with comparable results is shown.

 
Autocrine DC activation and regulation of DC IFN-{gamma}

So far we have demonstrated production of IFN-{gamma} by human DC on the single cell level and we have elucidated the role of the PRR TLR2 in this process. In the final part of this study we wanted to examine the regulation of DC-IFN-{gamma} and a possible role of this cytokine in autocrine DC activation based on a recently proposed so-called jump start model (19).

To investigate whether DC-IFN-{gamma} regulates its own expression, we stimulated DC with BCG in the presence of inhibitory Abs to IFN-{gamma}R1. However, secretion of DC-IFN-{gamma} remained essentially unchanged after blockade of the IFN-{gamma}R indicating that production of DC-IFN-{gamma} is not under autoregulatory control (Fig. 5A). We then wanted to know whether DC-IFN-{gamma} would be involved in the regulation of other cytokines released by DC after mycobacterial stimulation. As shown in Fig. 5B this was indeed the case as inhibition of the IFN-{gamma}R1 during BCG stimulation resulted in reduced production of TNF-{alpha}. Consequently, when we added exogenous recombinant IFN-{gamma} to BCG-stimulated DC, production of TNF-{alpha} 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 described to modulate IFN-{gamma} production in murine DC, lymphocytes, and macrophages. In our study these cytokines further induced BCG-mediated production of DC-IFN-{gamma}. Although single cytokines only moderately enhanced BCG-induced production of DC-IFN-{gamma}, IFN-{gamma} secretion was significantly up-regulated by additional stimulation with cytokine combinations (IL-12/IL-15 and IL-12/IL-18) (Fig. 6A). Stimulation of DC with cytokines alone for 24 h in the absence of BCG did not induce IFN-{gamma} production (Fig. 6A). However, low amounts of DC-IFN-{gamma} could be measured after prolonged (up to 3 days) stimulation of DC with cytokines alone (data not shown). Importantly, further up-regulation of BCG-induced DC-IFN-{gamma} by cytokines could not only be observed in culture supernatants of stimulated DC, but was also detectable on the single cell level by confocal microscopy (Fig. 6B).


Figure 5
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  		FIGURE 5. Autocrine IFN-{gamma} and BCG cooperate in the induction of cytokines. DC were generated from peripheral blood monocytes, purified by magnetic depletion of lymphocytes, and stimulated with BCG or left untreated. At 30 min before BCG treatment either a blocking Ab to IFN-{gamma}R1 ({alpha}IFN-{gamma}R1) or an isotype control (IgG1) were added (10 µg/ml). IFN-{gamma} (A) and TNF-{alpha} (B) in the culture supernatant were determined by ELISA. C, To examine the effect of exogenous IFN-{gamma} on BCG-induced cytokine release, DC were stimulated with a suboptimal dose of BCG (MOI = 0.5) or left unstimulated (iDC) and increasing doses of recombinant human IFN-{gamma} were added. TNF-{alpha} and IL-12 production were determined by ELISA. One representative experiment of six with comparable results is shown.

 

Figure 6
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  		FIGURE 6. IFN-{gamma} production by DC can be synergistically enhanced by a combined stimulation with BCG and IL-12/IL-15/IL-18. Purified DC were stimulated with a suboptimal dose of BCG (MOI = 0.5) or left untreated. IL-12, IL-15, or IL-18 (10 ng/ml each) were added in combination or alone as indicated. A, Supernatants were harvested 24 h past stimulation, and IFN-{gamma} content was determined by ELISA. B, Using some cultures of stimulated DC, cytospin preparations were prepared, immunostained (as for Fig. 3C) and analyzed by confocal microscopy. Note the enhanced signal for IFN-{gamma} in DC costimulated with BCG and cytokines. Isotype control stainings were negative and similar to Fig. 3C. One representative experiment of three with comparable results is shown.

 
Taken together the data of the final part of our study indicate that DC-IFN-{gamma} is part of a cytokine network secreted by human DC encountering mycobacterial pathogens. Within this network DC-IFN-{gamma} plays a role in autocrine DC activation as it regulates the production of other cytokines, namely TNF-{alpha}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IFN-{gamma} is a central mediator of cellular Th1 immunity and absolutely required for effective immunity against intracellular pathogens including mycobacteria. Thus, IFN-{gamma} gene disrupted mice are unable to control growth of M. tuberculosis and succumb to otherwise sublethal infectious doses of mycobacteria (12, 13). In addition, IFN-{gamma} also plays an important role in the activation of APC leading to both activation of antibacterial cellular responses and enhanced induction of antibacterial lymphocyte responses (36). Consequently, the question whether also APC themselves could produce IFN-{gamma} gained considerable interest (19).

After early reports on IFN-{gamma} production by human alveolar macrophages (15, 37) subsequent studies mostly focused on murine APC. Experiments with in vitro generated or ex vivo isolated murine APC demonstrated production of significant amounts of IFN-{gamma} by these cells (38, 39). Recently, there has also been increasing evidence for the production of IFN-{gamma} by myeloid cells in vivo based on experimental murine systems (20, 21). In a large number of these studies cytokines (mostly IL-12 and IL-18) and intracellular pathogens have been identified as potent inducers of DC-derived IFN-{gamma}. Based on these findings, a so-called jump start model for APC activation has been proposed (19).

Despite growing evidence for IFN-{gamma} production by murine myeloid cells until now virtually nothing is known about the potency of human APC to produce this important cytokine. Also, production of IFN-{gamma} by APC is still a matter of intensive debate because a significant number of studies did not report on production of IFN-{gamma} protein but rather focused on production of mRNA only (23). Moreover, most investigators reported on macrophages rather than DC and the contribution of contaminating lymphocytes has not always been clarified explicitly (22). Thus, this is the first study to provide a detailed analysis on the expression of IFN-{gamma} by human DC on the single cell level, to address the role of contaminating lymphocytes in this regard, and to elucidate some of the functional consequences of autocrine DC-IFN-{gamma} production. Using a cytokine secretion assay, in situ hybridization, and confocal microscopy we were able to detect mRNA and protein expression of IFN-{gamma} in human DC. In addition to pure morphology, identity of DC was confirmed by immunocytochemistry and the IFN-{gamma}-producing cells coexpressed DC-SIGN, a surface molecule specifically expressed on monocyte-derived DC and not present on lymphocytes or NK cells. Depending on the protocol for in vitro differentiation of monocyte-derived DC, starting cultures of monocytes can have a purity ranging from well below 80% up to almost 99% (40, 41, 42). In most of our experiments for standard DC differentiation, we obtained a DC purity between 90 and 97% with CD3-positive T cells being the most frequent contaminating cell population (see Fig. 2A, crude DC). Using a magnetic bead based Ab depletion system we succeeded in generating DC populations of high purity virtually devoid of contaminating lymphocytes and NK cells (see Fig. 2A, purified DC). Comparison of IFN-{gamma} production in cultures of crude and purified DC revealed higher cytokine levels in cultures of crude DC. Nevertheless, we consistently observed significant levels of IFN-{gamma} protein also in cultures of purified DC. Together with the single cell analysis by confocal microscopy these data clearly indicate that human DC have the capacity to produce IFN-{gamma} protein. At the same time these data also show that contaminating lymphocytes most likely contribute to the production of IFN-{gamma} in most cultures of conventionally generated monocyte-derived human DC. Parallel detection of IFN-{gamma} expression and BCG infection revealed a considerable proportion of DC, which were IFN-{gamma}-positive and BCG-negative. At first glance these findings suggest that direct infection is not absolutely essential for cytokine production. However, we have to consider that BCG uptake precedes cytokine production by many hours and mycobacteria might already be degraded in a proportion of IFN-{gamma}-positive DC.

After we had demonstrated production of IFN-{gamma} by human DC stimulated with BCG mycobacteria, we next wanted to elucidate some of the mechanistic and functional implications of this finding. TLR represents a family of ancient sensing molecules expressed on a variety of cell types and belong to a group of receptors commonly referred to as PRR (43). TLR2 has been identified as a major TLR mediating recognition of mycobacteria by immune cells. However, the relative contribution of different TLRs (namely TLR2 and TLR4) to antimycobacterial immunity under in vivo conditions remains controversial and might well depend on the mycobacterial strain used, the dose, and route of infection and the timing of the experiment (34, 44, 45, 46). Nevertheless, in vitro stimulation of murine myeloid cells with whole intact BCG mycobacteria seems to be preferentially mediated by TLR2 (33, 34, 44). Interestingly, extracts consisting of BCG cell wall skeleton and peptidoglycan extracted from BCG bacteria are dependent on both TLR2 and TLR4 signaling for activation of myeloid cells (47, 48). In our hands, TLR2 was clearly dominant over TLR4 in conferring cellular activation in response to BCG mycobacteria. HEK cells overexpressing TLR2 were strongly activated by BCG and this activation was fully comparable to the response achieved by the well-defined synthetic lipopeptide PAM3CysSK4. In contrast, no TLR4-dependent activation could be observed in HEK cells overexpressing TLR4. Similarly, blocking of TLR2 on human DC clearly reduced their cytokine production, and most important production of DC-IFN-{gamma}, in response to BCG. At the same time, blockade of TLR4 signaling did not substantially alter the immunological response of BCG-stimulated human DC. Given the complexity of the interaction of potential pathogen-associated molecular patterns on the surface of BCG mycobacteria with their receptors on human DC, it was not surprising that in subsequent experiments production of DC-IFN-{gamma} was not completely blocked in the presence of inhibitory Abs to TLR2. We would also like to note that stimulation of DC with the synthetic TLR2 ligand PAM3CysSK4 induced only low amounts of cytokines in DC except for IL-6 (data not shown). This finding is consistent with previous reports in which additional costimulation with IFN-{gamma} was required for full DC activation and IL-12 production (49). Combined, these data suggest that TLR2 is of crucial importance for the induction of DC-IFN-{gamma} by BCG mycobacteria, but additional receptors might also participate in this process. In addition, these experiments suggested to us the interesting possibility that DC-IFN-{gamma} could function as an autocrine amplifier and costimulator of TLR-mediated responses in human DC.

Thus, in the final part of our study we have investigated a possible autocrine amplification loop mediated by DC-IFN-{gamma}. Indeed, our experiments are consistent with such a process as inhibition of the IFN-{gamma}R resulted in reduced production of TNF-{alpha} after BCG stimulation. Interestingly, IFN-{gamma} does not regulate its own expression as blocking of IFN-{gamma}R did not change BCG-induced levels of DC-IFN-{gamma}. In the context of a possible autocrine IFN-{gamma}-driven APC activation model it is of note that for M. tuberculosis it has been described that the 19-kDa lipoprotein inhibits IFN-{gamma}-dependent activation of murine macrophages (50, 51) if prolonged TLR2 ligation precedes IFN-{gamma} signaling. It remains to be defined whether such effects are also effective in human professional APC such as DC or whether they are limited to macrophages, which are primarily involved in immediate host defense and pathogen elimination. Alternatively, these data offer the interesting possibility that virulent mycobacterial strains have developed mechanisms to circumvent autocrine APC activation by DC-IFN-{gamma} as a possible way to down-regulate or inhibit full immune activation.

A difference between murine and human APC was noted with regard to the effects of IFN-{gamma}-inducing cytokines. Although in murine macrophages and DC IL-12 alone or in combination with IL-18 has been described as a potent inducer of DC-derived IFN-{gamma} (18, 52), in our study in the absence of mycobacterial challenge, these cytokines induced human DC-IFN-{gamma} only after prolonged periods of stimulation (3 days). Nonetheless, IL-12, IL-15, and IL-18 and combinations thereof, were profound costimulators for induction of DC-IFN-{gamma} when combined with BCG stimulation.

In our study we have demonstrated for the first time production of the "lymphocytic" cytokine IFN-{gamma} by human DC. Recently, there has been accumulating evidence for the production of another lymphocytic cytokine, namely IL-2, by APC. Although initial studies focused on murine APC (53), IL-2 production has now also been observed in human DC. Interestingly, in human myeloid, DC production of IL-2 strongly depends on costimulation with IL-15 (54). Thus, our study supports growing evidence that certain cytokines like IL-2 and IFN-{gamma}, which have previously been thought to be exclusively produced by lymphocytes, might also be secreted by APC. In this regard we have no doubt that lymphocytes will produce higher amounts of IFN-{gamma} per cell compared with DC. Nonetheless production of even small amounts of lymphocytic cytokines by APC could still have a significant impact on immune responses in the respective micromilieu.

In conclusion, in this study we have described DC-derived IFN-{gamma} as a cytokine participating in a complex autocrine activation loop after mycobacterial stimulation. These findings might be of important relevance for the mechanistic comprehension of the early phases of immune responses against intracellular pathogens like mycobacteria.


   Acknowledgments
 
We especially thank Gabriele Bentien for brilliant technical assistance throughout the entire project. We thank Dr. Oliver Umland for the preparation of the IFN-{gamma} probe used for in situ hybridization, Erika Kaltenhäuser and Renate Bergmann for elutriation of monocytes and TNF-{alpha} ELISA, and Heike Kühl and Prof. E. Vollmer for support with in situ hybridization. We are grateful to Genentech for providing the anti-TLR2 Ab, we acknowledge the help of U. Wolfram (Miltenyi Biotec) with the cytokine secretion assay, and are grateful to Dr. Thomas Scholzen for skillful introduction into confocal microscopy techniques. Finally, we thank Prof. Stefan Ehlers for critical reading and helpful comments on the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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 and Project C8. Back

2 I.F. and D.M. contributed equally to this work. Back

3 Current address: H. Lee Moffitt Cancer Center, University of South Florida, Moffitt Research Center Building Room 2067, 12902 Magnolia Drive, Tampa, FL 33612. Back

4 Address correspondence and reprint requests to Dr. Sven Brandau, Division of Immunotherapy, Research Center Borstel, Parkallee 1-40, 23845 Borstel, Germany. E-mail address: sbrandau{at}fz-borstel.de Back

5 Abbreviations used in this paper: DC, dendritic cell; BCG, bacillus Calmette-Guérin; PRR, pattern recognition receptor; MOI, multiplicity of infection; HEK, human embryonic kidney. Back

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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