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The Journal of Immunology, 2002, 168: 6199-6207.
Copyright © 2002 by The American Association of Immunologists

A Novel Role for IL-3: Human Monocytes Cultured in the Presence of IL-3 and IL-4 Differentiate into Dendritic Cells That Produce Less IL-12 and Shift Th Cell Responses Toward a Th2 Cytokine Pattern1

Susanne Ebner2,*, Susanne Hofer*, Van Anh Nguyen*, Christina Fürhapter*, Manfred Herold{dagger}, Peter Fritsch*, Christine Heufler* and Nikolaus Romani*

Departments of * Dermatology and {dagger} Internal Medicine, University of Innsbruck, Innsbruck, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) derived from plasmacytoid precursors depend on IL-3 for survival and proliferation in culture, and they induce preferentially Th2 responses. Monocytes express not only GM-CSF receptors, but also IL-3Rs. Therefore, we examined whether IL-3 had an effect on the functional plasticity of human monocyte-derived DC generated in a cell culture system that is widely used in immunotherapy. DC were generated with IL-3 (instead of GM-CSF) and IL-4. Yields, maturation, phenotype (surface markers and Toll-like receptors), morphology, and immunostimulatory capacity were similar. Only CD1a was differentially expressed, being absent on IL-3-treated DC. In response to CD40 ligation DC generated in the presence of IL-3 secreted significantly less IL-12 p70 and more IL-10 compared with DC grown with GM-CSF. Coculture of naive allogeneic CD4+ T cells with DC generated in the presence of IL-3 induced T cells to produce significantly more IL-5 and IL-4 and less IFN-{gamma} compared with stimulation with DC generated with GM-CSF. These data extend the evidence that different cytokine environments during differentiation of monocyte-derived DC can modify their Th cell-inducing properties. A hitherto unrecognized effect of IL-3 on DC was defined, namely suppression of IL-12 secretion and a resulting shift from Th1 toward Th2.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)<3 are the most potent APC (1). Their interaction with naive T cells plays a key role in primary immune responses (2, 3). In particular, the interaction of T cells with DC is crucial for directing Th cell differentiation toward the Th1 or Th2 type. Several factors determine the direction of T cell polarization. The dose of Ag (4, 5), the strength of antigenic stimulation (6), the duration of TCR engagement (7), and the nature of costimulatory molecules (8) contribute to the balance of Th1 and Th2 responses. In addition, the cytokine microenvironment is important in Th cell differentiation toward the Th1 or Th2 cell type (9, 10, 11). Th1 and Th2 polarization is promoted by IL-12 and IL-4, respectively. IL-12 potently induces IFN-{gamma}-secreting Th1 cells (12). IL-12 is made by myeloid DC during maturation in response to microbial or T cell-derived stimuli (13, 14) and thereby induces Th1 responses. This is well established. In contrast, the contribution of DC to Th2 development still remains unclear.

In humans, DC derived from plasmacytoid precursors (DC2) do not produce IL-12 in response to CD40 ligation (15); they elicit Th2 responses, and their precursors (pDC2) are a major source of IFN-{alpha} (15, 16, 17). These lymphoid DC express different subsets of pattern recognition receptors to recognize different classes of Ags (18, 19). The regulation of T cell-mediated immune responses by DC of different ontogenetic pathways (myeloid DC/DC1 vs lymphoid DC/DC2) was called the evolutionary selection model by Liu et al. (20). The other, complementary model was termed the environmental instruction model, which means that each ontogenetically defined DC subset has a certain degree of flexibility in directing T cell responses (20). This depends on signals from pathogens and the microenvironment. For example, DC derived from plasmacytoid precursors (DC2) can elaborate IL-12 in response to stimulation via Toll-like receptor (TLR)9 (19) or IFN-{alpha} in response to virus (21). Thus, both the types of DC subsets as well as microenvironmental clues appear to be important for Th polarization.

IL-3 is the key cytokine for the generation of DC2 from high IL-3R-expressing plasmacytoid precursor cells (22). It ensures their proliferation and survival. Precursors for DC1 (i.e., monocytes) also express IL-3R, albeit at lower levels (15). We therefore wondered whether monocytes developing into DC would receive instruction signals via the IL-3R. Specifically, we asked whether IL-3 could alter the nature of a monocyte-derived DC as a typical Th1-inducing APC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and reagents

The culture medium used throughout was RPMI 1640 supplemented with 1% glutamine, 50 µg/ml gentamicin (all from PAA, Linz, Austria), 2-ME (Sigma-Aldrich, St. Louis, MO), and 1% autologous plasma. GM-CSF was purchased from Novartis (Basel, Switzerland; Leukomax; sp. act., 1.1 x 106 U/mg), IL-4 was obtained from Genzyme (Cambridge, MA; sp. act., 5 x 107 U/mg), and IL-3 was purchased from PeproTech (London, U.K.; sp. act., 1 x 107 U/mg). TNF-{alpha} (sp. act., 6 x 107 U/mg) was provided by Dr. G. R. Adolf (Bender, Vienna, Austria). IL-1{beta} (sp. act., 5 x 108 U/mg) and IL-6 (sp. act., 1 x 107 U/mg) were obtained from Genzyme, and PGE2 (prostine E) was purchased from Pharmacia & Upjohn (Buurs, Belgium). Abs used for flow cytometric analyses of the phenotype are listed in Table IGo.


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  		Table I. mAbs used for flow cytometry analyses

 
Generation of DC

DC were generated from adherent mononuclear cells in human blood according to established standard procedures (23, 24). Blood was freshly drawn from healthy volunteers at the local blood center. Monocytes were obtained by directly enriching for CD14+ cells by means of a magnetic sorting system (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany) or by depleting T cells by means of Ig-coated sheep erythrocytes (E-rosetting). Monocytes were seeded into six-well culture dishes at a density of 2 x 106 monocytes/well in 3 ml culture medium. The initial 7-day priming culture in the presence of GM-CSF (800 U/ml) and IL-4 (1000 U/ml) was followed by a 2-day differentiation culture in the additional presence of monocyte-conditioned medium or a defined cytokine mixture consisting of TNF-{alpha} (final concentration, 10 ng/ml), IL-1{beta} (10 ng/ml), IL-6 (1000 U/ml), and PGE2 (1 µg/ml) (25). GM-CSF and IL-4 were still present during this period. In the majority of experiments populations of immature DC were split in half on day 7 of culture. They were cultured for 2 more days in the presence or the absence of monocyte-conditioned medium or cytokine mixture. On day 9 cells were collected, and immature (i.e., those without monocyte-conditioned medium or without cytokine mixture) and mature (i.e., those with monocyte-conditioned medium or with cytokine mixture) DC were analyzed for cytokine production in parallel.

This culture system was modified such that GM-CSF was replaced by recombinant human IL-3 (10 ng/ml = 100 U/ml) throughout the initial 7-day priming culture and the 2-day differentiation culture. For reasons of simplicity these DC will hereafter be called IL-3 DC, as opposed to the standard DC (GM-CSF DC).

Stimulus to induce IL-12, IL-1{beta}, IL-10, and IL-8 production in DC

Murine myeloma cells transfected with the human CD154/CD40 ligand molecule (P3xTBA7 cells) were used to ligate the CD40 molecule on the surface of DC (26). Wild-type cells served as a negative control (P3x63Ag8.653-WT). These cells were a gift from Dr. R. A. Kroczek (Berlin, Germany).

Determination of DC-derived IL-12, IL-1{beta}, IL-10, and IL-8

Immature or mature DC were washed out (three times) of cytokine-containing culture medium. They were counted under the hemocytometer and analyzed for CD83 expression by flow cytometry, and 1 x 106 DC/ml were plated into 24- or 48-well multiwell tissue culture plates together with 0.5 x 106/ml transfected murine myeloma cells. Supernatants were taken at 48 h and were stored at -80°C until analysis by ELISA. Experiments were analyzed with a commercial IL-12 ELISA (BD PharMingen, San Diego, CA). The capture Abs used in this test specifically recognize the p70 heterodimer, but not the free p40 chains. Detection limits were 20 pg/ml IL-12. IL-1{beta}, IL-10, and IL-8 were analyzed with commercial ELISAs from CLB (Amsterdam, The Netherlands).

Purification of naive T cells and DC-T cell cocultures

PBMC were incubated with a mixture of mAbs, including CD14, HLA-DR, CD56, CD8, CD19, CD45RO, and CD40 (from BD PharMingen). Petri dishes were coated for 1 h with AffiniPure goat anti-mouse IgG (10 µg/ml; Jackson ImmunoResearch Laboratories, Avondale, PA). Naive CD4+CD45RA+ T cells were isolated using a panning technique described previously (27). This was repeated twice to obtain >95% pure CD4+ T cells. T cells were cocultured with allogeneic DC (GM-CSF DC vs IL-3 DC) in 24-well plates at a 4:1 ratio (1 x 106 T cells; 0.25 x 106 DC) for 6 days. Thereafter, T cells were restimulated with plate-bound anti-CD3 (5 µg/ml; BD PharMingen) and plate-bound anti-CD28 (1 µg/ml; BD PharMingen) for another 8 h (for FACS analyses) or 30 h (for ELISA analyses).

Determination of T cell-derived IFN-{gamma}, IL-4, IL-5, and IL-10

After 6 days of DC-T cell coculture, naive T cells were reactivated with plate-bound anti-CD3 and anti-CD28 for 30 h. Culture supernatants were frozen at -20°C until analysis by ELISA. IFN-{gamma} was determined with a commercial kit from BioSource (Nivelles, Belgium); IL-4, IL-5, and IL-10 production was measured with kits from CLB.

Flow cytometric detection of IFN-{gamma} and IL-4

After 6 days of DC-T cell coculture, T cells were reactivated with plate-bound anti-CD3 and anti-CD28 for 8 h. GolgiStop (1 µg/ml; BD PharMingen) was added to the cultures for 4 h before staining to prevent cytokine secretion. Cells were washed and stained according to the manufacturer’s protocols. All reagents were purchased from BD PharMingen (Cytofix/Cytoperm kit, R-PE-conjugated mouse anti-human IL-4 mAb, FITC-conjugated mouse anti-human IFN-{gamma} mAb).

Mixed leukocyte reaction

DC were gamma irradiated at 30 Gy, and graded doses were then added to 2 x 105 allogeneic T cells in 96-well, flat-bottom culture plates for 6 days. Proliferation was determined by the addition of 1 µCi [3H]thymidine (sp. act., 247.9 GBq/mmol = 6.7 Ci/mmol; New England Nuclear, Boston, MA) during the last 16 h of the culture period and subsequent measurment of incorporated radioactivity in a liquid scintillation counter (Wallac, Turku, Finland).

Quantitative determination of mRNA expression

Total RNA was isolated by TRIzol (Life Technologies, Vienna, Austria), and cDNAs were prepared with random primers (Superscript II RNase H-reverse transcriptase; Life Technologies). Quantitative PCR analysis was performed using real-time PCR (ABI PRISM 7700 sequence detector; Applied Biosystems, Vienna, Austria). Primer sequences for the detection of TLR mRNA were identical as described by Kadowaki et al. (18). Sequences for probes (FAM label) and primers specific for IFN-{alpha}1 mRNA (sense, 5'-cctcgccctttgctttactg; antisense, 5'-gcccagagagcagcttgact; probe, 5'-tggtcctggtggtgctcagctgc) and for DEC-205/CD205 mRNA (sense, 5'-ttcgatctcgcggagcc; antisense, 5'-gcacttgcccgtatttcca; probe, 5'-tctggccgcgcagctaatgacc) were selected using Primer Express software (Applied Biosystems). Subsets a and b of IFN-{alpha} are both amplified by the selected reagents. All primers and probes were synthesized by Microsynth (Balgach, Switzerland). For PCR, TaqMan PCR Master Mix from Applied Biosystems was used.

Determination of macropinocytosis and phagocytosis

The endocytic activity of GM-CSF DC and IL-3 DC was measured as described previously (28). FITC-dextran (Sigma-Aldrich) was used to measure mannose receptor-mediated endocytosis. Cells (105) were incubated with FITC-dextran (0.5 mg/ml) for 30 min at 37°C (control at 0°C) and then washed extensively with PBS containing 0.1% sodium azide. The samples were analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA). Data were analyzed using CellQuest software from BD Biosciences. Latex beads (0.5%, v/v; 2-µm diameter) for phagocytosis experiments were purchased from Seradyn (Indianapolis, IN). They were added to the cell cultures at a final dilution of 1/20.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human monocytes express CD116 (GM-CSF receptor {alpha}-chain) and CD123 (IL-3R {alpha}-chain)

We first examined the expression of two relevant cytokine receptors. In accordance with Rissoan et al. (15) we observed that the vast majority of freshly isolated monocytes (CD14+ cells) expressed both CD116, the GM-CSF receptor {alpha}-chain, and CD123, the IL-3R {alpha}-chain (data not shown). As suggested by their IL-3 sensitivity (22) and as directly measured by flow cytometry (15, 29), expression levels of CD123 on the surface of plasmacytoid precursors of DC (pDC2) were very high (~103–104 mean fluorescence intensity). No such highly CD123-positive subset was detected among the fresh monocyte population. Both receptors were expressed similarly at intermediate (~102) fluorescence intensities.

Morphology, yields, and endocytic capacity do not change markedly in response to IL-3

DC were cultured in GM-CSF and IL-4 or in IL-3 and IL-4 for 7 days. Preliminary experiments revealed that IL-4 could not be omitted from the cultures. Monocytes cultured in GM-CSF or IL-3 alone adhered for the most part and did not develop into DC. In the case of GM-CSF this was previously reported (30, 31). On day 7 DC were stimulated with cytokine mixture or monocyte-conditioned medium for 2 more days. These mature IL-3 DC cells showed grossly the same morphology as mature GM-CSF DC (Fig. 1Go). Immature IL-3 DC also were identical to immature GM-CSF DC (data not shown). We wondered whether DC grown in IL-3 would develop an extensive endoplasmic reticulum, as described for plasmacytoid DC precursors (22). Electron microscopy of immature and mature IL-3 DC did not reveal such structures however. The ultrastructural features of GM-CSF DC and IL-3 DC were similar.



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  		FIGURE 1. Morphology and yields of DC grown in the presence of IL-3. Upper panel, DC progenitors were primed for 7 days in the presence of IL-3 and IL-4, followed by a 2-day maturation period in the presence of cytokine mixture containing IL-1{beta}, IL-6, TNF-{alpha}, and PGE2. Note the numerous thin cytoplasmic processes (veils). Magnification, x550. Lower panel, Yields of immature and mature GM-CSF DC ({square}) and IL-3 DC ({blacksquare}). The number of monocytes plated on day 0 was set equal to 100%. Percentages indicate the numbers of recovered DC on day 9 of culture. Data are from 19 separate experiments; error bars indicate SDs.

 
For the determination of DC yields the number of monocytes plated on day 0 was set equal to 100%. Percentages indicate the numbers of recovered DC on day 7 or 9 of culture. Cell yields of immature GM-CSF DC and immature IL-3-DC on day 7 of culture were similar (data not shown). To be able to harvest immature and mature DC on the same day, immature DC were further cultured from days 7–9 in the presence of IL-3 and IL-4 but without maturation stimulus as previously described (13). Under these conditions, IL-3-containing cultures contained ~20% fewer cells than GM-CSF-containing cultures, as determined by counting in the hemocytometer with trypan blue (Fig. 1Go). No significant differences became obvious between the yields of mature GM-CSF DC and IL-3 DC (26 ± 11% (range, 7–48%) and 22 ± 11% (range, 7–47%), respectively; mean ± SD and ranges of 19 separate experiments).

The capacity to take up Ags was measured in two systems using FITC-dextran as an indicator of macropinocytosis and latex beads as an indicator of phagocytosis. In each case immature DC took up more of the material than mature DC. No differences became apparent between IL-3 DC and GM-CSF DC.

Immunostimulatory capacity in the mixed leukocyte reaction is similar

The ability to stimulate bulk T cells (Fig. 2GoA) as well as naive CD45RA+ T cells (Fig. 2GoB) was acquired during the maturation period induced by either monocyte-conditioned medium or cytokine mixture (25). The stimulatory capacity of IL-3 DC for T cells was comparable to that of GM-CSF DC.



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  		FIGURE 2. Immunostimulatory functions of immature and mature GM-CSF DC and IL-3 DC. A MLR was performed with both immature and mature GM-CSF DC and IL-3 DC as indicated. {square}, Immature GM-CSF DC; {blacksquare}, mature GM-CSF DC; {circ}, immature IL-3 DC; •, mature IL-3 DC. The maturation stimulus was a cytokine mixture containing IL-1{beta}, IL-6, TNF-{alpha}, and PGE2. Different numbers of DC were cocultured with 2 x 105 allogeneic bulk T cells (A) and CD45RA+ naive allogeneic T cells (B) for 6 days. Proliferation was measured by [3H]thymidine incorporation (cpm). The background proliferation of T cells alone was 355 cpm in A and 420 cpm in B. One representative experiment of three is shown.

 
Phenotypes of immature and mature DC cultured in GM-CSF plus IL-4 and IL-3 plus IL-4 differ only in CD1a expression

Monocytes were cultured in the presence of IL-4 and GM-CSF or IL-3 for 7 days, followed by another 2 days in the absence or the presence of the defined cytokine mixture as a maturation stimulus. The resulting populations of immature and mature DC were analyzed side-by-side by flow cytometry. The only distinct phenotypical difference was observed with CD1a. This surface marker showed some reduction in expression levels upon maturation when DC were cultured and matured in GM-CSF and IL-4, as previously described (24). In contrast, neither immature nor mature DC cultured in IL-3 and IL-4 showed any CD1a expression (Fig. 3Go).



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  		FIGURE 3. Comparative cytofluorographic analysis of GM-CSF DC and IL-3 DC. Lymphocyte-depleted PBMC were cultured for 7 days with either GM-CSF and IL-4 or IL-3 and IL-4, followed by a maturation period from day 7 to day 9 in the presence of a cytokine mixture containing IL-1{beta}, IL-6, TNF-{alpha}, and PGE2. Large cells (i.e., DC) were gated. The fluorescence of gated cells is depicted in the histograms. Thin lines in each panel represent staining with isotype-matched irrelevant control Igs. Each row of histograms represents FACS profiles of one individual side-by-side comparative experiment. Immature GM-CSF DC (left column) were compared with immature IL-3 DC (left middle column), and mature GM-CSF DC (right middle column) were compared with mature IL-3 DC (right column). The only differently expressed molecule is CD1a. IL-3 DC express no CD1a. Each marker was probed in at least three separate experiments.

 
All remaining markers tested (Table IGo) revealed no differences between GM-CSF DC and IL-3 DC. Fig. 3Go shows that immature IL-3 DC display the typical marker profile for immature DC. Like GM-CSF DC, they are CD86+/-CD83-CD14-CD11c+CD40+CD25-BDCA-4+BDCA-2-. A minor difference is seen in the expression of CD123, in that IL-3 DC express slightly more CD123 than GM-CSF DC. Also, expression of CD44, CD11a, CD11b, CD18, CD29, CD36, and CD49 a, b, c, d, e, and f, as well as CD205/DEC-205 and CD209/DC-SIGN is not different in GM-CSF DC and IL-3 DC (data not shown). Mature IL-3 DC show an expression profile of the analyzed molecules similar to mature GM-CSF DC (Fig. 3Go). Upon maturation of IL-3 DC the expression of CD83 and CD25 is induced, and the expression of CD86 and CD205/DEC-205 is enhanced. CD14 is low or absent. The expression of CD123, BDCA-4, CD11c, and CD40 is high, and BDCA-2 is absent. This pattern is identical with that of mature GM-CSF DC. Again, the expressions of CD44; CD11a; CD11b; CD18; CD29; CD36; CD49 a, b, c, d, e, and f; CD205/DEC-205; and CD209/DC-SIGN were identical in both populations (data not shown). The intracellular expression of the DC maturation marker CD208/DC-lysosome-associated membrane glycoprotein was assessed on cytocentrifuge specimens. Immature DC did not express this molecule, whereas mature DC yielded bright perinuclear staining for DC-lysosome-associated membrane glycoprotein. This was identical for GM-CSF DC and IL-3 DC (data not shown).

mRNA for pattern recognition receptors is not altered in DC cultured in IL-3

Expression of mRNA for TLR2, TLR4, TLR7, and TLR9 was determined by means of quantitative PCR. We consistently observed that mature DC expressed less receptor mRNA that immature DC.

TLR2 and TLR4 have been reported to be operative on human myeloid DC (DC1), whereas DC derived from plasmacytoid precursors (DC2) are equipped with receptors 7 and 9 (18). Monocyte-derived DC cultured in the presence of IL-3 displayed the same expression pattern as those cultured conventionally in the presence of GM-CSF (Fig. 4Go). They contained much mRNA for TLR2 and TLR4 and little, if any, mRNA for TLR7 and TLR9. Thus, IL-3 did not shift the TLR expression pattern toward a DC2 pattern.



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  		FIGURE 4. Expression of mRNA for TLR on DC. Real-time PCR analyses of four separate experiments (dotted, stippled, hatched, and filled bars, respectively) are shown. Immature (immat) and mature (mat) DC generated in the presence of GM-CSF and IL-4 (GM DC) are compared with DC generated in the presence of IL-3 and IL-4 (IL-3 DC). Note that the scales for the mRNA units on the y-axis differ by 1 log between the upper panels (TLR2 and TLR4) and the lower panels (TLR7 and TLR9). All DC populations tested express sizeable levels of mRNA for TLR2 and TLR4 (more so when immature) but very little mRNA for TLR7 and TLR9. IL-3 does not change this pattern. Mono, Freshly isolated control monocytes.

 
We also examined the lectin-like receptor CD205/DEC-205 and found the known up-regulation upon maturation, but no differences in mRNA expression between DC cultured in the presence of IL-3 or GM-CSF (data not shown).

IL-3 DC produce less IL-12 p70 and IL-1{beta} but more IL-10 in response to CD40 ligation

We have recently demonstrated that ligation of CD40 induces more IL-12 in immature/maturing than in terminally mature GM-CSF DC (13). We wondered whether IL-3 DC would show these same features and how they would compare to conventional monocyte-derived DC cultured in the presence of GM-CSF (GM-CSF DC). Not unexpectedly, the down-regulation of IL-12 p70 occurred in IL-3 DC just as described for GM-CSF DC (Fig. 5GoA).



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  		FIGURE 5. Cytokine production by CD40 ligand-stimulated DC. DC were cultured in the presence of either GM-CSF and IL-4 or IL-3 and IL-4 for 7 days, followed by a maturation period from day 7 to day 9 in the absence or the presence of a cytokine mixture containing IL-1{beta}, IL-6, TNF-{alpha}, and PGE2. The resulting immature and mature DC were plated at 1 x 106/ml in the presence or the absence of CD40 ligand-expressing cells for another 48 h, and supernatants were assayed for IL-12 p70, IL-1{beta}, IL-10, and IL-8. A, Both immature and mature IL-3 DC produce significantly less IL-12 p70 than GM-CSF DC. IL-12 values of individual side-by-side experiments are connected by lines (n = 14; p > 0.0001 for both immature and mature populations). B, Mature IL-3 DC produce significantly less IL-1{beta} (n = 5; p = 0.04), but more IL-10 (n = 5, not significant), than GM-CSF DC. Both subpopulations secrete equal amounts of IL-8 (n = 5).

 
More importantly, however, in all 14 independent experiments conducted, ligation of CD40 induced significantly less (p < 0.0001) IL-12 p70 production in IL-3 DC than in GM-CSF DC (Fig. 5GoA). This was true for both immature and mature IL-3 DC. All experiments were side-by-side comparisons. In eight of these experiments mature IL-3 DC secreted 10- to 50-fold less IL-12 p70 than the corresponding GM-CSF DC. We and others have previously shown that IL-4 enhances IL-12 production by conventional monocyte-derived DC (GM-CSF DC) (13, 32, 33). When IL-4 was present during the stimulation of GM-CSF DC with CD40 ligand-expressing cells, an increase in IL-12 secretion was observed. This increase was also visible in IL-3 DC, although it was not as pronounced (data not shown). CD40 ligand expressed on the transfectants induced DC maturation in both types of DC under the very conditions of stimulation for ELISA analyses (i.e., 1 x 106 DC in 1 ml for 48 h). This would rule out insufficient maturation of IL-3 DC as a cause of diminished IL-12 production.

We also measured IL-1{beta}, IL-10, and IL-8 secretion from mature (Fig. 5GoB) and immature (data not shown) DC populations that were stimulated by ligation of CD40. Compared with GM-CSF DC, IL-1{beta} was significantly reduced in mature IL-3 DC as well as in immature IL-3 DC. Mature IL-3 DC produced more IL-10 than GM-CSF DC. However, these differences were not significant. The amounts of IL-8 did not differ between GM-CSF DC and IL-3 DC regardless of their state of maturation.

IFN-{alpha} is the Th1-inducing cytokine for DC2 (20). It was studied by means of quantitative PCR analyses. Unstimulated DC cultured in the presence of either IL-3 or GM-CSF expressed similar levels of mRNA for this cytokine (data not shown).

IL-3 DC induce T cells to produce significantly more IL-5 and IL-4 and less IFN{gamma} compared with stimulation with GM-CSF DC

We next examined the nature of primary allogeneic T cell responses induced by GM-CSF DC and IL-3 DC. T cell-derived cytokines were measured by ELISA and intracellular FACS analyses. In eight independent experiments T cells cocultured with allogeneic mature GM-CSF DC secreted sizeable amounts of IFN-{gamma} (61–2000 ng/ml), but little IL-4 (14–28 pg/ml) and IL-5 (16–217 pg/ml; Fig. 6Go). In contrast, T cells cocultured side-by-side with allogeneic mature IL-3 DC secreted significantly less IFN-{gamma} (25–613 ng/ml; p = 0.0130), but significantly more IL-4 (34–303 pg/ml; p = 0.0143) and IL-5 (26–508 pg/ml; p = 0.0467). The polarized cytokine production profiles induced by GM-CSF DC and IL-3 DC were confirmed by the detection of intracellular cytokines by means of flow cytometry. As shown in a representative experiment in Fig. 7Go mature GM-CSF DC induced more IFN-{gamma}-producing cells than IL-4 (15 vs 2%), and mature IL-3 DC induced the inverse, namely more IL-4 than IFN-{gamma} (12 vs 2%). No differences in IL-10 secretion by T cells induced by GM-CSF DC vs IL-3 DC was evident (Fig. 6Go).



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  		FIGURE 6. GM-CSF DC and IL-3 DC influence the Th1 vs Th2 balance. Quantitation of Th1 and Th2 cytokines secreted by T cells and induced by mature GM-CSF DC and mature IL-3 DC. Human CD4+CD45RA+ naive T cells were cocultured with allogeneic mature GM-CSF DC and IL-3 DC. After 7 days of culture cells were restimulated for another 30 h with plate-bound anti-CD3 and anti-CD28 Abs. IFN-{gamma}, IL-4, IL-5, and IL-10 in culture supernatants were measured by ELISA (n = 7). IL-3 treatment of DC resulted in a shift of T cell responses toward a Th2 pattern, i.e., less IFN-{gamma} and more IL-4 and IL-5. Note that the IFN-{gamma} panel has a different y-axis (nanograms per milliliter).

 


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  		FIGURE 7. Two-color analysis of IL-4 and IFN-{gamma} expression by flow cytometry. After 6 days of DC-T cell coculture, T cells were restimulated with plate-bound anti-CD3 and anti-CD28 Abs for 8 h. Monensin (1 µg/ml) was added to the culture for the last 4 h before staining to prevent cytokine secretion. One representative experiment of 11 independent experiments is shown. DC cultured in the presence of IL-3 (IL-3 DC), instead of GM-CSF (GM-CSF DC), made T cells secrete more IL-4 but less IFN-{gamma} (compare horizontally). Quadrants were set according to the fluorescence intensities of FITC- and PE-conjugated isotype-matched control Igs with irrelevant specificity.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human monocyte-derived DC generated in the presence of GM-CSF and IL-4 (23, 24) induce Th1 responses by default (15). Here we report that monocyte-derived DC can be manipulated such that they secrete less IL-12 p70 and more IL-10 in response to a T cell stimulus (i.e., CD40 ligand). As a consequence, the induced Th cytokine secretion pattern shifts toward the Th2 phenotype. This can be achieved by simply replacing GM-CSF with IL-3 during the entire culture period. No such effect of IL-3 on DC was known hitherto. Our data extend and complement a recent report about the combined effects of IFN-{beta} and IL-3 (34) in that the instructive role of IL-3 was specifically delineated here.

Effects of IL-3 on DC

IL-3 was shown to be a survival-supporting and, to a lesser degree, proliferation-inducing cytokine for plasmacytoid precursors (15, 22). We demonstrate here that IL-3 exerts no such effect on the proliferation of precursors for DC1 (i.e., monocytes). Typically little, if any, cell proliferation occurs in cultures of monocytes in the presence of GM-CSF and IL-4. This was also true for cultures containing IL-3 and IL-4. A novel, instructive effect of IL-3 on monocyte-derived DC, however, becomes apparent from our experiments. IL-3 precludes the development of monocytes into DC that secrete the bioactive IL-12 p70 heterodimer in response to a T cell stimulus. A similar phenomenon has been described for other cell types, namely murine macrophages (35) and mast cells (36), but, to the best of our knowledge, not for monocyte-derived DC generated under conditions appropriate for clinical use, i.e., FCS-free and with the help of IL-4.

The instructive effect of IL-3 on plasmacytoid DC precursors (pDC2) seems to be somewhat different. When pDC2 are cultured in the presence of IL-3, they do not make IL-12 p70 and induce Th2 responses (15), similar to monocyte-derived DC generated in the presence of IL-3. If IL-3 is replaced by virus, these DC induce Th1 rather than Th2 responses. However, this is not achieved by IL-12, but by IFN-{alpha} production of DC (21). Thus, IL-3 in plasmacytoid DC precursors does not seem to act as a suppressor of differentiation toward potent IL-12-producing cells.

Plasticity of the Th cell-inducing potential of DC

The diverse functions of DC in immune regulation are determined by two different factors: their ontogenetic derivation (myeloid vs lymphoid DC) and the various instructive signals that they receive during innate immune responses from pathogens (37) and/or cytokines (20). Our data provide further evidence for the latter mechanism. With regard to phenotype and T cell stimulatory function, DC cultured in the presence of IL-3 are almost indistinguishable from conventionally cultured DC in the presence of GM-CSF, with the exception of CD1a expression. They do not acquire any of the phenotypical markers of DC2 such as BCDA-2 (38), TLR7, and TLR9 (18), or poor phagocytic and macropinocytic capacity (20). Thus, IL-3 does not appear to change the set of pathogens that monocyte-derived DC recognize. Yet, they induce different cytokine secretion patterns in T cells. Therefore, it seems that within a given ontogenetic lineage of DC cardinal features, such as pathogen receptor profiles, remain stable, but other important features, such as IL-12 production, may be subject to environmental (i.e., cytokines) influences. This emphasizes the idea that the evolutionary selection model and the environmental instruction model (20) are by no means mutually exclusive.

The IL-3 effect on monocyte-driven DC is underscored by relating it to recent data reported by Tanaka et al. (5). These authors showed that standard (i.e., GM-CSF and IL-4) monocyte-derived DC can induce T cells to make less IFN-{gamma} but more IL-4, IL-5, and IL-13 if used at low numbers and vice versa. During a primary MLR at a 1:4 DC:T cell ratio Th1 responses were induced. Despite using the same 1:4 ratio we observed increased Th2 cytokine production when DC had been cultured in IL-3.

Another recently proposed parameter involved in the Th1/Th2 decision is the early or late state of DC activation. According to this DC exhaustion model monocyte-derived DC produce IL-12 only during a short period after stimulation with LPS, thereby inducing Th1 differentiation. After the burst of IL-12 production, DC shut down IL-12 production and no longer polarize T cells toward the Th1 pathway but toward the Th2 pathway (39). In our experiments immature DC generated in the presence of IL-3 secreted less IL-12 than DC generated in the presence of GM-CSF. Upon maturation both IL-3 DC and GM-CSF DC down-regulated their IL-12 secretion similarly. This rules out that the reduced IL-12 p70 secretion by mature IL-3 DC is merely due to a more rapid exhaustion of these cells.

Phenotype: CD1a expression

The expression of only one phenotypical marker was altered on DC in response to IL-3. CD1a did not appear on the surface of DC that had been cultured in the presence of IL-3 compared with DC grown conventionally in the presence of GM-CSF or to monocytes cultured in the presence of GM-CSF (40). The expression of this molecule may indeed reflect some functional properties of DC. For instance, Chang et al. (41) cultured DC in a different culture medium (Yssel’s medium). The resulting DC lacked IL-12 production and drove the Th cell response toward a Th2 pattern. Similarly, Kalinski et al. (42) generated CD1a-low or -negative DC from monocytes in the continuous presence of PGE2. These DC made less IL-12 and more IL-10.

A functional implication of the lack of CD1a expression may relate to the function of CD1 molecules as efficient presenting molecules for microbial lipid Ags (43). This property may be reduced or absent in DC cultured in the presence of IL-3.

IL-10 production of DC

Mature IL-3 DC produce increased levels of IL-10 compared with mature GM-CSF DC following activation with CD40 ligand-expressing cells. Previous studies (13, 44) indicated that IL-10 inhibits IL-12 production by DC. This suggests that endogenously produced IL-10 may play a role in regulating the function of IL-3 DC in an autocrine fashion. Furthermore, IL-10 prevents cytokine synthesis (45) and accessory cell function (46) of monocytes and maturation of DC (47). Clearly, the IL-3 DC-derived IL-10 is not sufficient to prevent maturation, because these cells can fully mature and thereby develop a high T cell stimulatory capacity. It is also not sufficient to induce IL-10-producing regulatory T cells as described for murine pulmonary DC (48). Our data support the conclusion that the synthesis of IL-10 and IL-12 by DC is independently regulated, which is in line with studies demonstrating that, for instance, CD47 ligation on monocytes selectively inhibits their IL-12 production (49) or measles virus does the same with DC (50), both apparently independently of IL-10.

Quality of DC-induced Th cell responses

DC cultured in the presence of IL-3 induced Th cells that produced more IL-4 and IL-5 and less IFN-{gamma} than DC cultured in the presence of GM-CSF, indicating a shift toward a Th2 profile. An additional possibility for DC would be to induce regulatory T cells, as recently described by several groups (19, 51, 52). However, it is unlikely that IL-3-treated monocyte-derived DC would elicit regulatory T cells. In opposition to the work of Jonuleit et al. (52), mature IL-3 DC induced vigorous proliferation in allogeneic T cells. Furthermore, the resulting T cells did not secrete increased amounts of IL-10 as was also described (19, 51).

Methodical aspects: generation of monocyte-derived DC under the aegis of IL-3

We show here that it is possible to generate DC with IL-3 instead of GM-CSF. The resulting DC populations were similar in terms of cell yield, morphology, T cell stimulatory capacity, phenotype, and capacity to mature in response to a defined cytokine mixture (IL-1{beta}, IL-6, TNF-{alpha}, PGE2) (25). Like DC generated in the presence of GM-CSF (30, 31), the addition of IL-4 proved to be absolutely essential. It should also be emphasized that these cultures were performed free of FCS in the presence of 1% autologous plasma, i.e., in a culture system that is presently used for the generation of DC for adoptive transfer immunotherapy of tumors (53, 54, 55).

We considered the possibility that IL-3 did not act on the majority of monocytes (i.e., pDC1) but, rather, expanded a preexisting minor subpopulation of highly IL-3 receptive monocytes or pDC2. Several points argue against that possibility. 1) Flow cytometric analyses never revealed subsets of monocytes that would express high levels of IL-3R (CD123) as described for plasmacytoid DC precursors (pDC2) (15). 2) We never observed proliferating cells or dying cells in the DC cultures by phase contrast microscopy. Both phenomena would be expected if the majority of monocytes died, and only a small fraction of the cells survived and expanded. 3) Most importantly, IL-4, which was present in all cultures, was reported to effectively inhibit IL-3-dependent survival and proliferation of pDC2 (15).

Possible relevance in vitro and in vivo

Monocyte-derived DC were used as a model, because this cell type is now commonly applied for immunotherapy of cancers (54, 55). DC generated in the presence of IL-3 might have some relevance in immunotherapeutic approaches that aim at dampening hazardous Th1 responses such as those found in inflammatory autoimmune diseases. Such IL-3-treated DC may be expected to induce an immune deviation (56) from a Th1 to a Th2 cytokine secretion pattern, possibly resulting in the amelioration of clinical disease. These approaches, however, are still far from clinical application and need extensive further experimental testing.

In vivo, allergy-related cytokines and mediators may influence DC in allergic reactions in the skin. This has recently been shown in the case of histamine (57). In addition, mast cells, eosinophils, and T cells secrete IL-3 (58, 59, 60). This may reinforce or stabilize Th2 responses because DC that reach the lymph nodes from such inflammatory sites would preferentially elicit Th2 cells. Kadowaki et al. (21) have discussed this scenario for the natural IFN-{alpha}{beta}-producing cells, i.e., DC2, in allergic reactions. Our data allow this effect to be extended to myeloid DC (DC1).


   Acknowledgments
 
We thank colleagues who generously provided us with important reagents: R. A. Kroczek (Robert Koch Institute, Berlin, Germany), R. M. Steinman (Rockefeller University, New York, NY), S. Lebecque (Schering-Plough, Dardilly, France), Y. Van Kooyk (Nijmegen, The Netherlands), and N. A. Nicola (The Walter and Eliza Hall Institute, Melbourne, Australia). Furthermore, we thank Markus Forstner for technical help.


   Footnotes
 
1 This work was supported by grants from the Austrian Science Fund (P-14949 and P-12555-MED to N.R. and P-13794-MED to C.H.) and by the Tiroler Landeskrankenanstalten GmbH/Tyrolean Provincial Hospital Co. Back

2 Address correspondence and reprint requests to Dr. Susanne Ebner, Department of Dermatology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail address: susanne.ebner{at}uibk.ac.at Back

3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; IL-3 DC, monocyte-derived DC generated in the presence of IL-3 and IL-4; GM-CSF DC, monocyte-derived DC generated in the presence of GM-CSF and IL-4. Back

Received for publication February 7, 2002. Accepted for publication April 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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