of October 5, 2018.This information is current as IL-4

Maturation, and Is Further Enhanced byWhen the Stimulus Is Given at the Onset of

OptimalMonocyte-Derived Dendritic Cells Is Production of IL-12 by Human

Fritsch and Nikolaus RomaniA. Kroczek, Manfred Herold, Christine Heufler, Peter

RichardMatthias Schmuth, Angelika Weiss, Daniela Reider, Susanne Ebner, Gudrun Ratzinger, Beate Krösbacher,

http://www.jimmunol.org/content/166/1/633doi: 10.4049/jimmunol.166.1.633

2001; 166:633-641; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/1/633.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Production of IL-12 by Human Monocyte-Derived DendriticCells Is Optimal When the Stimulus Is Given at the Onset ofMaturation, and Is Further Enhanced by IL-4 1

Susanne Ebner,* Gudrun Ratzinger,* Beate Krosbacher,* Matthias Schmuth,*Angelika Weiss,* Daniela Reider,* Richard A. Kroczek,† Manfred Herold, ‡ Christine Heufler,*Peter Fritsch,* and Nikolaus Romani2*

Dendritic cells produce IL-12 both in response to microbial stimuli and to T cells, and can thus skew T cell reactivity toward aTh1 pattern. We investigated the capacity of dendritic cells to elaborate IL-12 with special regard to their state of maturation,different maturation stimuli, and its regulation by Th1/Th2-influencing cytokines. Monocyte-derived dendritic cells were gener-ated with GM-CSF and IL-4 for 7 days, followed by another 3 days6 monocyte-conditioned media, yielding mature (CD831/dendritic cell-lysosome-associated membrane glycoprotein1) and immature (CD832/dendritic cell-lysosome-associated membraneglycoprotein2) dendritic cells. These dendritic cells were stimulated for another 48 h, and IL-12 p70 was measured by ELISA. Wefound the following: 1) Immature dendritic cells stimulated with CD154/CD40 ligand or bacteria (both of which concurrently alsoinduced maturation) secreted always more IL-12 than already mature dendritic cells. Mature CD154-stimulated dendritic cellsstill made significant levels (up to 4 ng/ml). 2) Terminally mature skin-derived dendritic cells did not make any IL-12 in responseto these stimuli. 3) Appropriate maturation stimuli are required for IL-12 production: CD40 ligation and bacteria are sufficient;monocyte-conditioned media are not. 4) Unexpectedly, IL-4 markedly increased the amount of IL-12 produced by both immatureand mature dendritic cells, when present during stimulation. 5) IL-10 inhibited the production of IL-12. Our results, employinga cell culture system that is now being widely used in immunotherapy, extend prior data that IL-12 is produced most abundantlyby dendritic cells that are beginning to respond to maturation stimuli. Surprisingly, IL-12 is only elicited by select maturationstimuli, but can be markedly enhanced by the addition of the Th2 cytokine, IL-4. The Journal of Immunology,2001, 166:633–641.

D endritic cells are APCs specialized to initiate primaryimmune responses (1, 2). Several well-developed func-tional properties enable them to successfully fulfill this

task. The generation of immunogenic MHC/peptide complexesfrom protein Ags is efficiently done by immature dendritic cells.Signals delivered by inflammatory cytokines set off a maturationprocess whereby, in vivo, dendritic cells migrate to the T cell areasof draining lymphoid organs. There they present antigenic peptidesto naive T cells that are passing by in large numbers and select andbind the Ag-specific T cells from this circulating pool. The inter-actions of MHC/peptide and TCR and of costimulatory moleculeswith their counterreceptors lead to the activation of T cells that, inturn, results in their proliferation and cytokine synthesis. An ad-ditional crucial factor in the moment of dendritic cell/T cell inter-action in the lymphoid organ is the cytokine IL-12. This het-erodimeric cytokine (consisting of one p35 and p40 chain, each)

critically regulates the balance between Th1 and Th2 responses(3): IL-12 potently induces IFN-g-secreting Th1 cells.

Dendritic cells have repeatedly been shown to produce IL-12both in an unstimulated state (4) and, in much larger amounts,when stimulated by either bacteria or bacterial products (5, 6),virus (7), or by ligation of their CD40 and/or MHC class II mol-ecules (5, 8, 9). In the human system, this may only be true fordendritic cells of the myeloid lineage, so-called dendritic cells type1 (10). Dendritic cell-derived IL-12 was functional in that itskewed primary T cell responses toward a Th1 pattern (4, 11).Most studies hitherto, except one recent report (12), have not spe-cifically defined the maturational status of dendritic cells analyzed.This aspect is important, though, because it is the mature, T cell-activating dendritic cell in which IL-12 would presumably be mostrelevant for the generation of specific Th1 immunity. Thisprompted us to systematically study IL-12 production of dendriticcells as a function of their maturational state as well as of differentmaturation stimuli. In the light of recently reported feedback loopson IL-12 by Th2 cytokines (10), we have specifically investigatedthe influence of IL-4 and IL-10 on dendritic cell-derived IL-12. Weemphasized the study of monocyte-derived dendritic cells, a pop-ulation that is preferably used for immunotherapeutic approaches.

Materials and MethodsMedia and reagents

Culture medium used throughout was RPMI 1640 supplemented with 10%FCS (endotoxin,0.06 ng/ml), gentamicin (all obtained from PAA, Linz,Austria), and 2-ME (Sigma, St. Louis, MO). Alternatively, dendritic cellcultures were also set up in 1% autologous plasma, as described (13, 14).

Departments of *Dermatology and‡Internal Medicine, University of Innsbruck, Inns-bruck, Austria; and†Robert-Koch-Institut, Federal Health Administration, Berlin,Germany

Received for publication May 11, 2000. Accepted for publication September27, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants of the Austrian National Bank (Jubilaumsfonds6575) and the Austrian Science Fund (P-12555-Med) to N.R.2 Address correspondence and reprint requests to Dr. Nikolaus Romani, Departmentof Dermatology, University of Innsbruck, Anichstrasse 35, A-6020–Innsbruck,Austria.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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Generation of dendritic cells

Dendritic cells were generated from adherent mononuclear cells in humanblood according to established standard procedures (13, 14). Blood cellswere from freshly drawn blood or from buffy coats that were obtained fromthe local blood center. Briefly, an initial 7-day priming culture in the pres-ence of GM-CSF (800 U/ml) and IL-4 (1000 U/ml) was followed by a3-day differentiation culture in the additional presence of monocyte-con-ditioned medium (MCM).3 GM-CSF and IL-4 were still present during thisperiod. In the majority of experiments, populations of immature dendriticcells were split in half on day 7 of culture. They were cultured for 3 moredays in the presence or absence of MCM (13, 14). On day 10, cells werecollected, and immature (i.e., those without MCM) and mature (i.e., thosewith MCM) dendritic cells were analyzed for IL-12 production in parallel.Alternatively, in a few experiments, immature dendritic cells on day 7 ofculture were compared with mature dendritic cells on day 10 of culture,after maturation in the presence of MCM. Both types of immature dendriticcells were identical with regard to phenotype and IL-12 production. GM-CSF was obtained from Novartis (Basel, Switzerland; Leukomax, sp. act.,1.1 3 106 U/mg), and IL-4 was purchased from Genzyme (Cambridge,MA; sp. act., 53 107 U/mg). Alternatively, we used culture supernatant(5% v/v) of a cell line transfected with human IL-4 (IL-4-62) that wasprovided by Dr. A. Lanzavecchia (Basel, Switzerland).

Stimuli to induce and modulate IL-12 production indendritic cells

FixedStaphylococcus aureusCowan I strain (SACS, 10mg/ml Ig-bindingcapacity, Pansorbin cells, catalogue number 507861) was obtained fromCalbiochem (La Jolla, CA). Murine myeloma cells transfected with thehuman CD154/CD40 ligand molecule (P33 TBA7 cells) were used toligate the CD40 molecule on the surface of dendritic cells (15). Wild-typecells served as negative control (P33 63Ag8.653-WT). Alternatively, wecross-linked CD40 with anti-CD40 mAbs G28-5 (gift of Dr. E. Clark,Seattle, WA (16)) and MAB089 (Immunotech-Coulter, Marseille, France)as well as with total and ultracentrifuged culture supernatants of CD40ligand-transfected cells containing CD40 ligand bound to membrane frag-ments and soluble CD40 ligand, respectively (17). IL-10 (sp. act., 13 107

U/mg) was a gift of Dr. Ann O’Garra (DNAX Research Institute, Seattle,WA). IL-4 was from Genzyme (see above).

Determination of IL-12 production

Immature or mature dendritic cells were washed out (33) of cytokine-containing culture media. They were counted under the hemocytometer andanalyzed for CD83 expression by flow cytometry, and 13 106 dendriticcells/ml were plated into 24-well or 48-well multiwell tissue culture platesin total volumes of 1 and 0.5 ml of culture medium, respectively. (mAbHB-15a, anti-CD83 was a gift of Dr. Thomas F. Tedder, Durham, NC;FITC-conjugated anti-CD83 was from Coulter-Immunotech, Marseille,France.) Supernatants were taken at 48 and 72 h and stored at280°C untilanalysis by ELISA. For most experiments, we used a sandwich ELISA,which was generously provided by Drs. D. H. Presky and M. K. Gatelyfrom Hoffmann-LaRoche (Nutley, NJ; capture mAb, 20C2; detection mAb,peroxidase-conjugated 4D6). The exact protocol has been described pre-viously (18, 19). Few experiments were analyzed by means of a commer-cial IL-12 ELISA (Quantikine; R&D Systems, Minneapolis, MN). Thecapture Abs used in both tests specifically recognize the p70 heterodimer,but not the free p40 chains. Detection limits were 20 pg/ml of IL-12.

Flow cytometric detection of IL-12 production

PE-conjugated mouse mAb C11.5, directed against the p40 subunit of hu-man IL-12, was used to stain saponin-permeabilized cell populations thathad been stimulated for 30 h with CD40 ligand-expressing cells in thepresence (last 5 h) of brefeldin A to achieve some accumulation of cytokinewithin the cell (20). All reagents and the staining protocol were from BDPharMingen (San Diego, CA).

Other methods

Human IL-12 p40 and p35 mRNA was detected by PCR and liquid hy-bridization, as described previously (21). IFN-g was measured with a com-mercial ELISA (BioSource-Medgenix, Fleurus, Belgium). Binding of mAbdendritic cell-lysosome-associated membrane glycoprotein (DC-LAMP)(mouse IgG1) (22) on acetone-fixed cytospins was visualized by a biotin-

ylated anti-mouse Ig (Amersham-Pharmacia, Amersham, U.K.), followedby Texas Red-conjugated streptavidin (Amersham); after blocking of re-sidual binding sites with an excess of mouseg-globulin (100mg/ml), den-dritic cells were counterstained with an FITC-conjugated anti-HLA-DRmAb (clone L243; BD PharMingen, San Jose, CA). DC-LAMP was a giftof Dr. Serge Lebecque, Laboratory for Immunological Research, Schering-Plough (Dardilly, France). Neutralizing mAbs against human IFN-g (cloneB27) and isotype-matched control Abs were purchased from BD Phar-Mingen and used at final concentrations of 20mg/ml.

ResultsIn preliminary experiments, we found no difference in IL-12 pro-tein levels between 48- and 72-h incubation periods. Therefore, the48-h time point was used for all further ELISA analyses. IL-12 p70heterodimer secreted by unstimulated dendritic cells (4) was al-most always below the threshold of detectability of the ELISA. Asanother preliminary, CD40 expression was comparatively assessedon immature and mature dendritic cells and found to be similarlyhigh on both populations (Fig. 1). This contrasted with the expres-sion of CD83 that clearly distinguished the two maturational states(Figs. 1 and 2A). In addition, the up-regulated expression of CD86,the lack of CD115 expression, and a pronounced veiled morphol-ogy under phase contrast were used as markers for mature den-dritic cells (data not shown). It should be emphasized in this workthat we use “immature dendritic cell” as the term to describe amonocyte differentiated for 6–7 days in the presence of GM-CSFand IL-4 (23, 24). It is clearly more advanced than the early (25)immature dendritic cell such as a Langerhans cell in situ, in that ithas already sizeable levels of surface MHC class II molecules andit expresses high levels of CD40. Yet, it still requires maturationstimuli to further differentiate and to acquire those features thatcharacterize the terminally mature dendritic cell (13, 14): 1) noreversion back to a macrophage, 2) greatly augmented T cell-stim-ulatory capacity for MLR and CTL, and 3) de novo expression ofmarkers such as CD83 or DC-LAMP (22). These features are sim-ilar to those of dendritic cells that matured spontaneously fromblood after 2 days of culture (26) or of cultured epidermal Lang-erhans cells (27, 28).

3 Abbreviations used in this paper: MCM, monocyte-conditioned medium; DC-LAMP, dendritic cell-lysosome-associated membrane glycoprotein; SACS, fixedStaphylococcus aureusCowan I strain.

FIGURE 1. Immature and mature dendritic cells express comparablelevels of CD40. Dendritic cells were cultured in the presence or absence ofMCM from day 7 to day 10. The resulting immature (left) and mature(right) populations were stained with mAb G28-5, anti-CD40 (lower row).For comparison, the degree of maturation is demonstrated by the expres-sion of CD83: immature dendritic cells are largely negative (upper left);mature dendritic cells uniformly express high levels of CD83 (upper right).Histograms show CD40 or CD83 fluorescence of large cells (bold line),gated as depicted in the dot plots in Fig. 2. Dotted line shows staining withisotype-matched control Ig.

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Staphylococci induce more IL-12 in immature than in maturemonocyte-derived dendritic cells

In 11 independent experiments, IL-12 values from immature dendriticcells ranged between 2.1 and 0.02 ng/ml; values from correspondingpopulations of mature dendritic cells ranged from 0.5 to 0 (i.e., belowdetection threshold) ng/ml. However, in all experiments, immature

dendritic cells produced higher levels than mature dendritic cells (Fig.3). It is of note that in 7 of 11 populations of mature dendritic cells,IL-12 in the supernatants was below the level of detection, i.e.,,20pg/ml. Analysis of data by means of the two-samplet test showed thatthe differences between immature and mature dendritic cells werestatistically significant (p , 0.05).

FIGURE 2. Dendritic cells express CD83 and DC-LAMP upon stimulation with bacteria or ligation of CD40. Dendritic cells were cultured in thepresence or absence of MCM from day 7 to day 10. The resulting immature (left) and mature (right) populations were plated at 13 106 cells/ml in thepresence or absence of potential IL-12 stimuli (SACS, CD40 ligand-expressing cells, MCM) for another 48 h (from d10 to d12). Supernatants were assayedfor IL-12 by ELISA, and cells were analyzed by flow cytometry (A) or immunohistochemistry (B).A, Histograms show CD83 fluorescence of large cells(bold line), gated as depicted in the dot plots for the cells on day 10. Dotted line shows staining with isotype-matched control Ig. Theleft paneldemonstratesthat CD83 is induced on immature dendritic cells by bacteria (SACS), ligation of CD40 (CD40-L), and MCM to similar degrees. Theright panelshowsthat mature dendritic cells retain CD83 expression for 48 h even in the absence of any stimuli (compare d10 with d12/none).B, Fluorescence panels showdouble labeling for DC-LAMP (red fluorescence) and HLA-DR (green fluorescence). Note that no DC-LAMP-positive cells can be found when cells werecultured in the absence of CD40 ligand-expressing cells (left). In contrast, coculture with CD40 ligand transfectants leads to the up-regulation of DC-LAMPin virtually all HLA-DR-positive cells, i.e., dendritic cells, irrespective of the presence (right) or absence (middle) of 500 U/ml IL-4. HLA-DR-negativecells are CD40 ligand-expressing cells. Magnification,3400.

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SACS has been described as a potent maturation stimulus fordendritic cells (14). FACS analyses revealed that also under thevery conditions of the IL-12 assays (i.e., 13 106 cells/ml, 48-hincubation), maturation occurred as detected by the induction ofCD83 expression (Fig. 2A).

Ligation of CD40 induces more IL-12 in immature than inmature monocyte-derived dendritic cells

Initially, we tested the conditions for cross-linking CD40 by meansof CD40 ligand-expressing cells (TBA7 cells). CD40 ligand ex-pression of TBA7 cells was high; it was clearly more than thelevels reported for activated T cells (data not shown). MaximalIL-12 release by dendritic cells was achieved at a ratio of oneTBA7 cell to two dendritic cells. This ratio was kept for all addi-tional experiments. When proliferation of TBA7 cells was stoppedby irradiation with 15–30 Gy from a Cs source, they elicited con-siderably lower amounts of IL-12 from dendritic cells (data notshown). Therefore, viable TBA7 cells were used in all assays.

In 11 independent experiments, IL-12 values from immaturedendritic cells ranged from.100 to 1.26 ng/ml; values from cor-responding populations of mature dendritic cells were between 29and 0.11 ng/ml. Like with SACS stimulation, immature dendriticcells produced higher levels than mature dendritic cells in all ex-periments (Fig. 4). However, in contrast to SACS stimulation, pop-ulations of mature dendritic cells did elaborate substantial amountsof IL-12 in all experiments, the lowest concentration measuredbeing 110 pg/ml. Analysis of data by means of the two-samplettest showed that the differences between immature and mature den-dritic cells were statistically significant (p , 0.05). These datawere confirmed with dendritic cells that had been cultured in thepresence of 1% autologous human plasma (n 5 22) rather than10% FCS, as in the experiments reported above (Fig. 4). IL-12production in plasma-supplemented cultures was lower than inFCS-containing media, though. The down-regulation of IL-12 pro-duction upon maturation of dendritic cells did not only occur whenMCM was used as a maturation stimulus, but also when a definedcytokine cocktail consisting of IL-1b, IL-6, TNF-a, and PGE2 (29)was employed: Experiment 1, 19 ng/ml in immature vs 1.3 ng/ml

in mature dendritic cells; experiment 2, 1 vs 0.5 ng/ml. Stimulationof dendritic cells with control wild-type cells was consistently neg-ative. Addition of a mAb against the CD40 ligand completely ab-rogated IL-12 induction (data not shown).

Ligation of CD40 can induce maturation of dendritic cells (5).FACS analyses revealed that also under the very conditions of theIL-12 assays (i.e., 13 106 cells/ml, 48-h incubation), maturationoccurred in the presence of CD40 ligand-expressing cells, as de-tected by the expression of CD83 (Fig. 2A) as well as of intracel-lular DC-LAMP (22) (Fig. 2B). It is also noteworthy that CD83 ofalready mature dendritic cells (on day 10) remained stably on thesurface during the 2-day duration of the stimulation culture, evenin controls in which no stimulus was added (Fig. 2A).

Ligation of CD40 by anti-CD40 mAbs yielded inconsistent re-sults. mAb G28-5 did not induce IL-12 in some experiments; insome it did. In those experiments, the same phenomenon was not-ed: immature dendritic cells made more IL-12 than mature den-dritic cells in response to the Ab (immature vs mature dendriticcells, IL-12 p70 in pg/ml: Expt. 1,.500 vs 52; Expt. 2, 456 vs 46).MAB089 never induced IL-12. Likewise, total and ultracentri-fuged culture supernatants of CD40 ligand-transfected cells, con-taining CD40 ligand bound to membrane fragments and solubleCD40 ligand, respectively (17), were inactive in our assays.

Finally, we wished to test the possibility that immature and ma-ture dendritic cells made similar amounts of IL-12, but that pop-ulations of mature dendritic cells contained proteases that wouldefficiently degrade the cytokine. Therefore, rIL-12 was added toimmature and mature dendritic cells and incubated for 48 h. Su-pernatants analyzed by ELISA showed no substantial degradationof IL-12 in either population (data not shown).

Fully mature skin-derived dendritic cells make no detectableIL-12

Dendritic cells emigrated from whole skin explants were alsotested for their ability to produce bioactive IL-12. These cells area mixture consisting of epidermal Langerhans cells and dermaldendritic cells. All dendritic cells within these populations werefully mature, as previously shown (30, 31), and as monitored bymorphology under phase contrast and by CD83 and CD86 expres-sion (data not shown). In three independent experiments, SACS

FIGURE 3. Immature dendritic cells stimulated by bacteria producemore IL-12 than mature dendritic cells. Dendritic cells were cultured in thepresence or absence of MCM from day 7 to day 10. The resulting immatureand mature populations were plated at 13 106 cells/ml in the presence orabsence of SACS for another 48 h, and supernatants were assayed for IL-12by ELISA. Values of individual experiments are connected by lines. Al-though there is considerable variability between the individual experi-ments, each experiment shows that immature dendritic cells make moreIL-12 than mature dendritic cells.

FIGURE 4. Immature dendritic cells stimulated by ligation of CD40produce more IL-12 than mature dendritic cells. Dendritic cells were cul-tured in the absence or presence of MCM from day 7 to day 10. Theresulting immature and mature populations were plated at 13 106 cells/mlin the presence or absence of CD40 ligand-expressing cells for another48 h, and supernatants were assayed for IL-12 by ELISA.Left, Eexperi-ments conducted in FCS-containing medium, andright, experiments inmedium containing autologous plasma. Values of each individual experi-ment are connected by a line. Although there is considerable variabilitybetween the individual experiments, each experiment shows that immaturedendritic cells make more IL-12. Note that mature dendritic cells still se-crete substantial amounts of IL-12, particularly in FCS-containing medium.

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did not induce any measurable IL-12 p70. In two different exper-iments using CD40 ligand-transfected cells as stimulus, IL-12 p70production was also below the threshold of detection.

MCM induces maturation without IL-12 production

Because the stimuli that brought about IL-12 production (CD40ligation and SACS) also induced maturation, we wonderedwhether MCM, the classical stimulus for maturation (13, 14, 32),would also do so. Immature dendritic cells on day 7 or 10 wereassayed for IL-12 in the presence or absence of MCM. In sixindependent experiments, virtually no IL-12 was induced byMCM; however, dendritic cells from parallel cultures that werestimulated with CD40 ligand or SACS did elaborate the cytokine(Fig. 5). FACS analyses (Fig. 2A) proved that MCM rendered den-dritic cells stably mature also under the specific culture conditionsused for collecting supernatants for ELISA (i.e., 13 106 cells/ml;48 h).

It should be noted that in supernatants from standard maturationcultures (i.e., from day 7–10 in six-well plates at a cell density of0.73 105 cells/ml in the presence of 33% MCM), IL-12 was foundonly in 4 of 47 cultures, albeit at a low concentration.

Comparison of IL-12-inducing stimuli

From Figs. 1 and 4 it can be read that CD40 ligation is the stron-gest of the three stimuli tested. This becomes more apparent whenthe same data are plotted as side-by-side comparisons within dif-ferent individual experiments (Fig. 5). When CD40-induced IL-12production of immature dendritic cells is set equal to 100%, SACSelicits on average about one-fifth of this amount (19.96 37%;range 0.1–139%;n 5 16). MCM induce only very little IL-12(4.4 6 6, 9%; range 0.1–17, 9%;n 5 6).

IL-12 production at the single cell level

Intracellular FACS staining of CD40 ligand-stimulated dendriticcell populations using a mAb against the p40 subunit of IL-12showed unequivocally that IL-12 had accumulated in CD831 cells(Fig. 6). This indicated that IL-12 synthesis and maturation hadproceeded simultaneously. It also means that the high levels ofIL-12 are not produced by immature, but rather by maturing den-dritic cells. FACS analyses also confirmed that already maturedendritic cells made less IL-12 than maturing dendritic cells.

Differential production of IL-12 is transcriptionally regulated

Next we examined whether the differential secretion of IL-12 het-erodimer protein in immature and mature dendritic cell was due to

de novo synthesis. To this end, the expression of mRNA for thep35 and p40 subunits of IL-12 was investigated. A semiquantita-tive PCR analysis revealed that, in response to ligation of CD40,immature dendritic cells expressed more mRNA for both p35 andp40 than mature dendritic cells (Fig. 7). This was not as pro-nounced when SACS was used as a stimulus for IL-12 production.

Interferon-g

IFN-g has been described as necessary costimulus for IL-12 pro-duction by dendritic cells (33). Therefore, this cytokine was mea-sured in parallel with the IL-12 assays. In a series of 13 indepen-dent experiments (in medium containing autologous plasma), a

FIGURE 5. CD40 ligation by CD40 ligand-transfected cells is the morepowerful stimulus for IL-12 production. Data from Figs. 1 and 4 are ar-ranged in a different manner to highlight the comparison between CD40ligation, bacteria (SACS), and MCM. Dendritic cells were cultured in theabsence or presence of MCM from day 7 to day 10. The resulting immatureand mature populations were plated at 13 106 cells/ml in the presence orabsence of CD40 ligand-expressing cells or SACS or MCM for another48 h, and supernatants were assayed for IL-12 by ELISA. Values of indi-vidual experiments are connected by lines. Horizontal line indicates thedetection limit of the IL-12p70 ELISA. Note that the scales on they-axesare differently graded.

FIGURE 6. IL-12 is synthesized in maturing dendritic cells. Immatureand mature dendritic cells on day 10 were cocultured with CD40 ligand-expressing cells (right) for 30 h, the last 5 h thereof in the presence ofbrefeldin A to stop cytokine secretion. Dendritic cells in the absence of theCD40 stimulus (left) do not show p40 staining. Note that those cells thatexpress IL-12 p40 are CD831, i.e., they have matured in response to CD40stimulation. Mature dendritic cells (lower right) make low amounts of p40.

FIGURE 7. Immature dendritic cells stimulated by ligation of CD40express more mRNA for both IL-12 chains than mature dendritic cells.Dendritic cells were cultured in the presence or absence of MCM from day7 to day 10. The resulting mature and immature populations, respectively,were plated at 13 106 cells/ml in the presence or absence of CD40 ligand-expressing cells and SACS for another 18 h, and cell lysates were subjectedto PCR analyses. Note that both the mRNAs for p35 (40 cycles;left) andp40 (35 cycles;right) are up-regulated in response to the stimuli applied(compare with “DC only”). mRNA levels in response to CD40 ligation arehigher in immature than in mature dendritic cells (compare arrowed lanes).Positive control,top panels,far right lanes.

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significant (r 5 0.81) positive correlation between the values ofIL-12 and IFN-g became apparent (data not shown): Most culturesof CD40-stimulated immature dendritic cells contained moreIFN-g (maximum,.60 ng/ml IFN-g) than cultures of CD40-stim-ulated mature cells (maximum, 9.2 ng/ml IFN-g). Few experi-ments with SACS-stimulated dendritic cells revealed the same cor-relation (data not shown). It was not further investigated whetherthis cytokine was produced by dendritic cells or by few contami-nating T cells. NK cells were ruled out as producers of IFN-g: Infive independent FACS analyses, we detected virtually no CD561

cells, i.e., NK cells.

IL-4 enhances IL-12 production by monocyte-derived dendriticcells

When IL-4 was present during the stimulation of dendritic cellswith CD40 ligand-expressing cells, a marked increase of IL-12secretion was observed. Up to almost the 10-fold amount of IL-12was induced by IL-4. This observation was made both in FCS-containing cultures (Fig. 8) and in cultures with 1% autologousplasma: 698, 108, 995, 961, 139, 546, and 356% for immaturedendritic cells in seven experiments, and 125, 438, 183, and 243%for mature dendritic cells in four experiments; IL-12 production inthe absence of IL-4 was set equal to 100%. Thus, IL-4 enhancedIL-12 production irrespective of the state of dendritic cell matu-ration. IL-4 did not alter the degree of maturation of dendritic cells,as determined by morphology under phase contrast, CD83 expres-sion by FACS (data not shown), and DC-LAMP expression oncytospins (Fig. 2B). In a series of five independent experiments, wefound that IL-4 did not lead to increased amounts of IFN-g in thecultures, but to clearly enhanced levels of IL-12 (698, 108, 995,961, and 139%). Conversely, the neutralization of IFN-g with amAb did not prevent the IL-4-induced augmentation of IL-12 (Ta-ble I).

IL-10 inhibits IL-12 production by monocyte-derived dendriticcells

IL-10 was shown to inhibit IL-12 synthesis in murine dendriticcells (8). In this study, we investigated the effects of IL-10 onCD40-induced IL-12 production in human dendritic cells. In sixindependent experiments, a concentration of 10 U/ml (i.e., 1 ng/ml) IL-10 did not consistently inhibit IL-12 production. When 100U/ml (i.e., 10 ng/ml) IL-10 was present during the 48-h stimulationwith CD40 ligand, a clear-cut reduction of IL-12 secretion wasobserved (Fig. 8). The mean reduction with 100 U/ml IL-10 was61% for populations of immature dendritic cells, and 66% for ma-ture dendritic cells. Thus, dendritic cells at both states of matura-tion were inhibited to a similar degree by the high dose of IL-10.

DiscussionIn this study, we further dissect the intricate regulation of IL-12 asa function of dendritic cell maturation (immature vs maturing vsterminally mature; see below). We find that human dendritic cellsproduce the bioactive IL-12 p70 heterodimer most abundantlywhen they begin to respond to certain maturation stimuli. Theyreduce this powerful capacity as maturation proceeds, as describedrecently (12). We extend these data in several regards, the mostimportant ones being that 1) surprisingly, the Th2 cytokine IL-4markedly enhances IL-12 synthesis and secretion by both imma-ture and mature dendritic cells; 2) this phenomenon occurs also inthe clinically relevant culture system with autologous plasma (in-stead of FCS); 3) down-regulation of IL-12 production is also ob-served with skin-derived dendritic cells (complementing one re-cent report dealing specifically with Langerhans cells (34)); and 4)only select maturation stimuli (CD40, bacteria) can induce IL-12production in dendritic cells.

IL-12 p70 production is not a general feature of dendritic cellmaturation

We observed that CD40 ligation and (less though) bacteria inducedmassive IL-12 p70 production in immature dendritic cells. In con-trast, MCM, the classical maturation stimulus (13, 14), did notinduce substantial IL-12 production when applied in an identicalexperimental setting as the other stimuli (nor did the combinationof TNF-a and PGE2 in two experiments). This latter combinationwas reported to induce IL-12 p40 secretion, though (35). Thus, ofthe three stimuli tested in this study, two induced maturation andIL-12 secretion (CD40 ligation and bacteria), whereas one (MCM)led to maturation without concomitant IL-12 secretion. This is sim-ilar to the findings of Cella et al. (5), who noted IL-12 p70 induc-tion in dendritic cells only with CD40 ligation and to some degreewith viral infection (7), but not with other stimuli such as LPS orTNF-a. The discrepancy with regard to bacterially induced IL-12(in this study, good IL-12 induction; Cella et al. (5), no IL-12induction) may be due to different reagents (staphylococci, FCS).In vivo it would be advantageous if a dendritic cell made the potentcytokine IL-12 only if threatened by microbes or if in physicaltouch with T cells, rather than in response to any inflammatorycytokine milieu.

Relevance of maturation-linked IL-12 production capacity:immature dendritic cells

In vivo (e.g., in the epidermis), dendritic cells receive maturationand migration stimuli by inflammatory cytokines (36), often in theabsence of microbes. In that case, IL-12 is needed only when den-dritic cells have arrived in the lymph nodes and interact with Tcells. Maturation in the presence of MCM may be regarded as anin vitro equivalent for this case. In a scenario in which microbes

FIGURE 8. Effects of IL-4 and IL-10 on the IL-12 production of den-dritic cells. Dendritic cells were cultured in the absence or presence ofMCM from day 7 to day 10. The resulting immature and mature popula-tions were stimulated with CD40 ligand-expressing cells in the presence orabsence of IL-4 (50 and 500 U/ml) or IL-10 (10 or 100 U/ml) for another48 h, and supernatants were assayed for IL-12 by ELISA. IL-12 productionin the absence of IL-4 or IL-10 was set equal to 100% (dashed line). Notethat the absolute values for this production are much higher for immature(16.2 ng/ml;n 5 7) than for mature (1.4 ng/ml;n 5 7) dendritic cells. IL-4leads to an increase in IL-12; it even augments the already high IL-12production of immature dendritic cells. IL-10 inhibits consistently only atthe high dose.

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are present, high levels of bacterially induced dendritic cell-de-rived IL-12 could be beneficial in that they would contribute to theinflammation (activation of NK cells, maintenance and enhance-ment of Th1 state of infiltrating Th cells, and, as a consequence,macrophage activation) and thus help with the clearance of micro-organisms. Inflammation (e.g., in the skin) might be further fueledby high levels of IL-12 derived from the interaction of CD40-expressing migrating dendritic cells that encounter effector ormemory T cells expressing CD40 ligand. In vivo examples under-score our in vitro data: Dendritic cells in the spleen of mice ex-posed in vivo toToxoplasmaAgs respond with vigorous IL-12production (6). The initial strong IL-12 immunostaining was pre-dominantly found at the edge of the T cell area, but also in theperiarteriolar region, implying that these dendritic cells were notfully mature in situ. Similarly,Leishmania-infected dendritic cellsin the spleen produce IL-12 in situ (37).

Relevance of maturation-linked IL-12 production capacity:mature dendritic cells

Upon encounter with Ag-specific T cells in the T cell areas oflymphoid organs, IL-12 is crucial for the establishment of a Th1response. When a dendritic cell that arrives in the lymph nodevia the afferent lymphatics finds and binds an Ag-specific T cellin the T areas (38), it signals to the resting naive T cell via itsMHC/peptide complexes (signal 1) and costimulatory mole-cules (signal 2). T cell activation ensues and within a period offew hours, activated T cells up-regulate CD154 (CD40 ligand)expression. CD154, in turn, engages with CD40 on the surfaceof the dendritic cell and now signals flow in the inverse direc-tion: The T cell induces terminal maturation (i.e., further up-regulation of costimulatory molecules) and IL-12 production inthe dendritic cells (39, 40). This determines the default pathwayof dendritic cells to induce Th1 responses. Probably in the mi-croenvironment of the intracellular spaces in lymph nodes orspleen, small quantities of IL-12 might suffice to reach biolog-ically active concentrations. It should be emphasized that IL-12production was found to be markedly down-regulated in maturedendritic cells; it did not completely disappear, though. Sub-stantial quantities (up to some hundred pg/ml) were still madeby mature dendritic cells in most experiments. Higher concen-trations of IL-12 may even be harmful.

Augmenting effects of IL-4: mechanism

The presence of IL-4 during the stimulation period strongly increasedthe levels of IL-12 in response to CD40 ligation. The reason for thissomewhat unexpected finding may be a previous conditioning of den-

dritic cells during the 7-day culture in the continuous presence of IL-4,perhaps similar, but clearly not identical with what was described byD’Andrea et al. (41) as priming: These authors had observed anincreased IL-12 production by IL-4-pretreated PBMCs in response tobacteria. We observed an increased IL-12 production by IL-4-pre-treated dendritic cells in response to CD40 ligation plus IL-4. Thus,the otherwise IL-12-inhibiting cytokine IL-4 turned out to be IL-12enhancing when the cells had been pretreated (conditioned) with IL-4.From very recent data by Hochrein et al. (42), who used dendritic cellsthat had been generated in the absence of IL-4, it appears that theIL-12-enhancing effect of IL-4 does not depend on a prior exposure tothe same cytokine. A similar observation was made with CD40 li-gand-stimulated murine dendritic cell-containing populations by Tak-enaka et al. (43). IFN-g appears not to mediate the IL-4 effect, becauseits production was not induced by IL-4 in our hands, and moreover,IL-4 has typically been described to inhibit IFN-g rather than enhanc-ing it (44, 45). In addition, neutralization of IFN-g in the stimulationassays did not prevent the IL-4-induced increase in IL-12 production.This is in line with a recent report by Kalinski et al. (46), who ob-served the same phenomenon induced by IL-4 derived from a Th2clone that was deficient in IFN-g production. IL-4 also does not act byinfluencing the maturation status of dendritic cells: maturation mark-ers CD83 and DC-LAMP were up-regulated in response to CD40ligation, irrespective of IL-4 treatment. Although IL-4 can augmentIL-12 production in such a potent way, it is not a prerequisite for thelarge amounts of IL-12 made by dendritic cells. This was first con-cluded by Cella et al. (5), who showed high levels of CD40-inducedIL-12 in freshly isolated dendritic cells that had never encounteredIL-4 in vitro. It is underscored by the data of Koch et al. (8): murinespleen dendritic cells were induced to make large amounts of IL-12p70 by cross-linking with anti-CD40 mAb. These dendritic cells alsonever had contact with IL-4 during their generation.

Augmenting effects of IL-4: relevance

When exogenous IL-4 is added to cocultures of APCs and T cells,a Th2 response (i.e., IL-4-producing T cells) is the consequence(47). If a Th2 response, e.g., to fungal hyphae (48) or to helminthicparasites (49), occurs in the environment of a lymph node, onemight expect that the resulting T cell-derived IL-4 would skew allother ongoing or beginning immune responses toward a Th2 pat-tern. Our data would indicate that there may be some balancingmechanism ensuring that Th1 responses are not necessarily sup-pressed in an IL-4-rich milieu. This finding seems important forimmunotherapy (see below). While our work was in the finalstages of review, Hochrein et al. (42) reported that IL-4, and evenmore so IL-4 plus IFN-g, enhance the IL-12 p70 production of

Table I. Neutralization IFN-g does not abrogate the IL-4-enhanced IL-12 p70 production by CD40-stimulated dendritic cells

Expt. No.

Immature Dendritic Cellsa Mature Dendritic Cellsa

No IL-4 500 U/ml IL-4 No IL-4 500 U/ml IL-4

AControl mAbb

BAnti-IFN-g

CControl mAbb

DAnti-IFN-g

EControl mAbb

FAnti-IFN-g

GControl mAbb

HAnti-IFN-g

1 1280c 1280 1590 1410 480 410 790 7402 1600 1550 8630 8420 890 800 1100 10003 140 200 290 460 60 50 210 240

a Dendritic cells were cultured in medium containing autologous plasma until day 7 in the presence of GM-CSF and IL-4 and further on until day 10 either with GM-CSF1IL-4 (immature dendritic cells) or additionally with a MCM as a maturation stimulus (mature dendritic cells). These populations were then stimulated via ligation of CD40 usingCD40 ligand-expressing cell for another 48 h. Supernatants were analyzed by the IL-12 p70-specific ELISA. IL-4 was present or not during these 48 h.

b IL-12 p70 values in the absence of CD40 ligation were below the threshold of detection and are therefore not listed in the table. Values in the absence of Ab (data not shownhere) or in the presence of the isotype- and concentration-matched control Ab were similar.

c All IL-12 values are given in pg/ml. Note that, firstly, immature dendritic cells make more IL-12 than mature ones as also shown in Fig. 4 (columns A vs E); secondly,IL-4 enhances CD40-induced IL-12 production of both immature and mature dendritic cells as also shown in Fig. 8 (columns A vs C, and E vs G); and thirdly, the neutralizationof IFN-g during the stimulation period does not prevent the increase in IL-12 (columns C vs D, and G vs H).

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murine and human dendritic cells, and Kalınski et al. (46) foundthat IL-4 secreted by Th2 cells mediates high level IL-12 p70production by immature human dendritic cells. We confirm thesedata and extend them in that we show the effect of IL-4 in anFCS-free culture system relevant for adoptive immunotherapy andfor populations of mature monocyte-derived dendritic cells, i.e.,those dendritic cells that are able to prime naive T cells and arepreferentially used in immunotherapy (50, 51).

Inhibitory effects of IL-10

The inhibition of IL-12 synthesis by IL-10 in mature dendritic cellswas also somewhat unexpected. Steinbrink et al. (52) and Thurneret al. (53) have demonstrated that mature dendritic cells are resis-tant to the effects of IL-10 in the MLR. Three explanations areconceivable. First, it is possible that the dose of 100 U/ml (i.e., 10ng/ml) of IL-10 is unphysiologically high and already toxic. Wehave not further explored this possibility, except for simple trypanblue staining of cell populations at the end of the 48-h IL-12 assay.However, by this criterium, no IL-10-induced toxicity was de-tected. The low dose of IL-10 (1 ng/ml) did not consistently inhibitmature dendritic cells, the average inhibition being222%. Sec-ond, one may assume that the amounts of IL-12 that were mea-sured in populations of mature dendritic cells were derived fromfew, still immature or maturing dendritic cells that were still sus-ceptible to inhibition by IL-10. Third, IL-10 may have differentialeffects on mature dendritic cells. We observed in this study that thesame dose of 100 U/ml of IL-10 did not affect the phenotypical(CD83, CD86 expression) and morphological (nonadherence,veils) characteristics of mature dendritic cells, whereas in parallelcultures it inhibited IL-12 secretion, as described. This might shiftthe Th1/Th2 balance in ensuing T cell responses toward Th2. Nei-ther IL-12 nor the IFN-g/IL-4 balance of resulting T cell respond-ers was measured in previous work pinpointing the stability ofmature dendritic cells (52, 53). This hypothesis is underscored byour previous finding with a population of classically mature den-dritic cells, namely mouse spleen dendritic cells (8): IL-10 totallyblocked IL-12 p70 secretion. When mature spleen dendritic cellswere used to repetitively stimulate allogeneic T cells, the presenceof IL-10 led to the development of a Th2 pattern of T cell cyto-kines (F. Koch, personal communication).

Significance for clinical immunotherapy

Dendritic cells have been widely used and (successfully) tested inanimal models of tumor therapy, and a number of clinical trials arecurrently running (e.g., 51, 54, 55). Monocyte-derived mature den-dritic cells are often used as a convenient source of large numbersof human dendritic cells (53). Three sets of data from our exper-iments may be of relevance in a clinical setting: 1) Our finding thatmature dendritic cells were less responsive to CD40 ligation (i.e.,T cell interaction) in terms of IL-12 production seems counterpro-ductive at first glance. Yet, it is likely that the small amounts ofIL-12 still produced by mature dendritic cells will suffice for Th1skewing within the microenvironment of the lymph nodes. 2) Ourobservation that IL-10 inhibits CD40-induced IL-12 production indendritic cells should alert us that under circumstances of highIL-10 levels in the body, e.g., in tumor situations (56), dendriticcell therapies might be impaired (57) and might need adjuvanttreatment such as cytokines. Induction of anergy in melanoma-specific CTL by IL-10-pretreated immature (i.e., during the mat-uration culture) dendritic cells was recently demonstrated (58). 3)Finally and most importantly, the fact that dendritic cells makemuch more IL-12 when IL-4 is present seems encouraging forstrategies in which a predominant Th1 response is desired, forexample, therapy of tumors or microbial infections. It seems con-

ceivable that Ag-pulsed dendritic cells that arrive in a lymph nodewith an IL-4-rich milieu (e.g., atopic state) would still be able toskew a T cell response toward a Th1 pattern, perhaps even better.Additionally, IL-4 may allow for the development of Th2 mech-anisms that also appear to be critically involved in tumor immunity(59, 60), without inhibiting therapeutically administered dendriticcells.

AcknowledgmentsWe thank Susanne Neyer for generously providing her practical expertiseand skills in molecular biology, Hella Stossel for immunocytochemistry,Karin Salzmann for help with ELISAs, and Dr. Franz Koch for criticaldiscussions.

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