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The Journal of Immunology, 2002, 168: 3793-3800.
Copyright © 2002 by The American Association of Immunologists

Suppression of IL-12 Production by Soluble CD40 Ligand: Evidence for Involvement of the p44/42 Mitogen-Activated Protein Kinase Pathway1

Miriam Wittmann2, Petra Kienlin, Susanne Mommert, Alexander Kapp and Thomas Werfel

Department of Dermatology and Allergology, Hannover Medical University, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-12 is a key cytokine in skewing immune responses toward Th1-like reactions. Human monocytes/macrophages produce high amounts of bioactive IL-12 when a priming signal (IFN-{gamma} or GM-CSF) precedes a second signal (e.g., LPS). We and others have previously shown that preincubation with LPS before this stimulation procedure can efficiently and selectively suppress the production of IL-12 by human monocytes. In this study, we show that an almost complete suppression of IL-12 production can also be observed after preincubation of monocytes with costimulatory cell surface molecules that bind to members of the TNFR superfamily (CD40 ligand, TNF-related activation-induced cytokine (TRANCE)). The suppression of IL-12 was observable on the mRNA and protein levels and was not due to endogenous production of known IL-12 antagonists (i.e., IL-10, IL-4, and PGE2), to an increased number of cells undergoing apoptosis, nor to down-regulation of the IFN-{gamma} or CD40 receptor. Cell surface expression of the costimulatory molecules CD80 and CD86 was not reduced by the preincubation procedure, and only a moderate reduction of IL-6 production was observed. Several studies have identified signal transduction pathways that are activated by CD40 signaling, including activation of mitogen-activated protein kinases. The presence of the extracellular signal-related kinase-specific mitogen-activated protein kinase kinase 1/2-specific inhibitors PD98059 and U0126 abrogated suppression induced by sCD40 ligand or other second signals. This indicates that activation of extracellular signal-regulated kinase 1/2 contributes to the underlying mechanism of IL-12 suppression. This mechanism may be relevant in other inflammatory responses and may help to develop therapeutic strategies in Th1-mediated diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several members of the TNFR superfamily play critical roles in the initiation and regulation of the immune response (1). These structurally related proteins modulate a wide variety of cellular functions (i.e., proliferation, differentiation, and cell survival) and thus critically influence the course of inflammatory responses.

CD40, a cell surface glycoprotein expressed on monocytes, dendritic cells, B cells, and some other cell types, is a member of the TNFR superfamily (2) that conditions professional APCs for efficient specific T cell stimulation. The ligation of CD40 by CD40 ligand (CD40L3 or CD154), which is transiently expressed on activated T cells (3, 4), induces a marked up-regulation of adhesion and costimulatory molecules on APC (5) which improves their APC function, resulting in enhanced T cell stimulatory capacity (6). The CD40-CD40L costimulatory pathway has been shown to play a crucial role in humoral responses in humans to induce the production of various monokines (7) and, in particular, to be one of the major regulators of IL-12 induction by cells of the monocyte/macrophage lineage (6, 8, 9).

TNF-related activation-induced cytokine (TRANCE), also called receptor activator of NF-{kappa}B ligand (RANKL), has been shown to be expressed on activated T cells (10). The interaction between receptor activator of NF-{kappa}B (RANK), another member of the TNFR superfamily expressed on human APC, and TRANCE has been described to cooperate with CD40L in its functions on dendritic cells (11), including enhanced production of IL-12 (12).

IL-12, composed of disulfide-linked p40 and p35 chains, is a central immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses (13, 14). Although IL-12p35 is expressed more ubiquitously, IL-12p40 expression appears to be restricted to cell types that express biologically active IL-12 and is strongly inducible by LPS and CD40 ligation in certain cell types (9, 15). The available data suggest that IL-12-inducing stimuli act via enhancement of IL-12p40 promoter activity (16, 17, 18). Because of its critical role in determining a Th1/Th2 balance, elucidating the mechanism of IL-12 production during Th cell-APC interactions seems important. It is well documented that a priming signal (i.e., IFN-{gamma} or GM-CSF) is indispensable for the production of high amounts of bioactive IL-12 (18, 19, 20), which is produced upon stimulation with the challenging or second signal (i.e., bacterial products, including LPS, bacterial DNA, lipoteichonic acid, heat shock proteins, and intracellular parasites). In contrast, a wide variety of factors have been described which are able to down-/counter-regulate IL-12 production, including IL-10, PGE2, IL-4, vitamin D, histamine, and signaling through complement receptors (21, 22, 23, 24, 25, 26, 27, 28).

The molecular mechanisms underlying both IL-12 induction and suppression are not fully understood. It has been suggested by different authors that mitogen-activated protein (MAP) kinases (MAPK) are involved in IL-12 regulation in APCs. The MAPK are a group of protein serine/threonine kinases that are activated in response to a variety of extracellular stimuli. The major subgroups of MAPKs comprise the extracellular signal-regulated kinases (ERKs), the c-Jun amino-terminal kinases, and the p38 MAPKs. Activation of the p38 pathway has been shown to be involved in IL-12 p40 promoter activity and cytokine release in mouse APC (29, 30, 31) and, very recently, also in human monocyte-derived dendritic cells (32, 33), whereas there are some data indicating that activation of the ERK pathway acts to suppress IL-12 secretion (29, 31, 32). However, the targets of p38 or ERK1/2 that mediate IL-12 p40 expression/suppression have not yet been identified.

In this study, we demonstrate, by means of inhibitors that selectively target the ERK MAPK signaling cascades, that CD40 engagement seems to induce ERK activation in unprimed human monocytes, resulting in suppression of IL-12. We present data showing that the timing of stimuli encountered is a crucial component in regulating IL-12 production in which activation of the ERK pathway seems to be critically involved.


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

All cytokines were used as purified recombinant human preparations. Human IFN-{gamma} and TNF-{alpha} were obtained from R&D Systems (Wiesbaden, Germany), LPS was derived from Escherichia coli serotype 055:B5 (Sigma-Aldrich, Deisenhofen, Germany). Anti-IL-4 Ab useful for neutralization of human IL-4 bioactivity (R&D Systems) and neutralizing rat anti-human IL-10 mAb (BD PharMingen, Hamburg, Germany) were provided with a low endotoxin level. Indomethacin was purchased from Serva (Braunschweig, Germany); soluble CD40L (sCD40L) and RANKL were obtained from Alexis (Günberg, Germany). The endotoxin content of all reagents was determined by the Limulus amebocyte lysate assay and was <350 pg/ml for the concentrations used in cell culture. The MEK1/2-specific inhibitor U0126 was obtained from New England Biolabs (Frankfurt, Germany), PD98059 and SB202474 (a negative control for PD98059) were obtained from Calbiochem (Schwalbach, Germany).

Cell isolation and culture

PBMC from healthy donors were separated by Ficoll-Hypaque density gradient centrifugation and resuspended in IMEM (Biochrom, Berlin, Germany) supplemented with 4% human AB serum. Monocytes were purified by centrifugal counterflow elutriation. The resulting cell preparations contained up to 90% monocytes as assessed by CD14 staining (FITC-conjugated anti-CD14; Coulter-Immunotech, Hamburg, Germany) and FACS analysis. The normal preincubation time was 16 h; stimulation was performed using the priming signal IFN-{gamma} (300 U/ml), followed 2 h later by the second signal (50 ng/ml LPS or 2 µg/ml sCD40L), as described previously (34).

Messenger RNA isolation and reverse transcription (RT)

mRNA was isolated from 105 monocytes using a mRNA isolation kit (Roche, Mannheim, Germany) according to the supplier’s instructions. For RT-PCR analysis, RNA was then subjected to first-strand cDNA synthesis using oligo(dT)15 for full-length cDNA synthesis. The RT reaction mixture (20 µl) contained final concentrations of 50 U Expand-RT (Roche), 10 mM DTT, 1x first-strand RT buffer for Expand-RT, 0.5 mM of each dNTP (Roche), RNase inhibitor (Life Technologies, Gaithersburg, MD), and 80 pmol oligo(dT)15 (Roche).

Real-time fluorescence PCR

Real-time fluorescence PCR was performed using the LightCycler (Roche). For quantitative PCR the dsDNA binding dye SYBR Green (LightCycler-FastStart DNA Master SYBR Green I Kit; Roche) was used according to the supplier’s instructions. PCR was performed by rapid cycling in a reaction volume of 20 µl with 0.5 µM of each primer (IL-12p40 and {beta}-actin as previously described (34)) and 1 µl cDNA. Two microliters of LightCycler-FastStart DNA Master SYBR Green I (containing buffer, FastStart Taq DNA polymerase, dNTPs (with dUTP instead of dTTP), 10 mM MgCl2, a calibrated amount of SYBR Green I dye (Roche), and an additional 1.4 µl MgCl2 (final concentration, 2.75 mM) were used. After an initial denaturation step at 95°C for 360 s, amplification was performed using 35 cycles of denaturation (95°C), annealing (54°C for both IL-12p40 and {beta}-actin), and extension (72°C). Fluorescence was measured at the end of the annealing period of each cycle (for IL-12p40 fluorescence was acquired at 84°C to exclude primer dimers from the quantification, which were found in the water/medium control in some reactions) to monitor amplification. Real-time monitoring of the amplification allows quantification of the samples during the log-linear phase of the PCR. For quantitative analysis the second derivative maximum method was used (35). A six-point standard (100, 20, 10, 2, and 1%) was run with every PCR using dilutions of the corresponding positive control. Undiluted standard was defined as 100%; a standard curve and the relative amount of target in the unknown samples were calculated using the LightCycler software. After amplification was complete, a final melting curve was recorded by cooling the samples at 20°C/s to 65°C and then increasing the temperature to 95°C at 0.2°C/s. Fluorescence was measured continuously during the slow temperature rise to monitor dissociation of the PCR product. Thus, each specific PCR product gives rise to a product-specific melting peak (for {beta}-actin, 90°C; melting peak for p40, 88.2°C), which was verified by conventional agarose gel electrophoresis to correspond to amplified fragments of 225 bp ({beta}-actin) and 290 bp (IL-12p40), respectively. The entire process took <30 min, with no separate manipulation of the product necessary.

Flow cytometric analysis of intracellular cytokines and membrane molecules

Intracellular staining and quantification of cytokines were conducted as previously described (34). During the stimulation procedure brefeldin (Sigma-Aldrich) was added at 3 µg/ml. Cells were harvested, washed twice in PBS, then fixed with 4% ice-cold phosphate-buffered paraformaldehyde for 15 min at 4°C, and washed in PBS. To facilitate diffusion of the Ab through the cell membranes, cells were permeabilized in PBS with 0.1% saponin (Riedel de Haen, Seelze, Germany) for 15 min. Thereafter, pretitrated cytokine-specific mAb diluted in the permeabilization buffer (PBS-saponin) were added and incubated for 45 min at 4°C. The PE-conjugated cytokine-specific mAb and IgG1 isotype control mAb were used at a final concentration of 2 µg/ml (monoclonal mouse anti-human-IL-12 (p40/p70); this Ab reacts with human IL-12p40 monomer and with the p70 heterodimer, but not with the p35 monomer; monoclonal rat anti-human IL-6; BD PharMingen, San Diego, CA). After subsequent washings in permeabilization buffer, cells were resuspended and measured in PBS by flow cytometric analysis.

Expression of surface Ags was assessed using the following PE- or FITC-labeled monoclonal mouse anti-human Abs: CD80, CD86, CD14, and CD40L (Coulter-Immunotech, Hamburg, Germany); CD40 (Alexis); and IFN-{gamma}R Ab (a gift from R. D. Schreiber, Washington University School of Medicine, St. Louis, MO). FITC-labeled annexin V (Alexis) was used at a final concentration of 250 ng/ml, and propidium iodide (Mobi-Tec, Göttingen, Germany) was used at a final concentration of 1 µg/ml. Samples were analyzed on a FACScan flow cytometer (BD Biosciences, Heidelberg, Germany). Results were analyzed using CellQuest software (BD Biosciences).

Western blot analysis

Adherence-purified monocytes (2 x 106) were lysed at different time points after stimulation with sCD40L, IFN-{gamma}, or IFN-{gamma} plus LPS (usually 15 min after addition of the stimulus). Total cell lysates were obtained in 50 mM Tris (pH 6.8), 1% SDS, 15% glycerol, and 4 M urea. Fifteen microliters were removed for protein determination before the addition of 2 µl {beta}-ME and 10% bromophenol blue/sample, respectively. Proteins from the cell lysates (50–100 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Protean BA85, Schleicher & Schuell, Dassel, Germany). ERK1/2, p38 MAPK, and Stat1 phosphorylation were assessed using PhosphoPlus p44/42 MAPK (Thr202/Tyr204), PhosphoPlus p38, and PhosphoPlus Stat1 (Tyr701) Ab kit, respectively, according to the manufacturer’s instructions (New England Biolabs, Frankfurt am Main, Germany). Molecular weight marker and positive controls were probed along with the samples. The signals were detected by ECL after incubation with the appropriate secondary Abs.

Protein determination

Protein contents were determined for all Western blot samples using the Bio-Rad protein assay (Munich, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Souble CD40L inhibits the induction of IL-12 production in human monocytes

High amounts of IL-12 are only produced by human APC upon stimulation with a priming signal (i.e., IFN-{gamma} or GM-CSF) followed by a second signal (e.g., LPS) (11, 12, 13, 21). As shown in Fig. 1Go sCD40L acts as an excellent second signal, resulting in high IL-12 production by IFN-{gamma}-primed human monocytes. However, preincubation with sCD40L before stimulation with a priming and a second signal very efficiently down-regulates IL-12 production in the same cells (Fig. 1Go). A similar suppression of IL-12 could also be observed using TNF-{alpha} (36) or sRANKL during the preincubation; for all these mediators the suppression of IL-12 production was >75% and was dose dependent. Time kinetic experiments revealed that the preincubation time necessary for IL-12 suppression by sCD40L was at least 4 h (Fig. 2Go), pointing to an active mechanism of IL-12 suppression. Preincubation with the second signal did not result in a general down-regulation of all monocyte functions, as CD86/CD80 surface expression was not reduced under the same experimental settings (Fig. 3Go). The mean fluorescence intensity for CD86 was 797 ± 243 (±SEM) for IFN-{gamma}/LPS-stimulated monocytes compared with 742 ± 274 for monocytes preincubated with 2 µg/ml sCD40L before stimulation with IFN-{gamma}/LPS (n = 5). IL-6, which was determined along with IL-12, declined upon CD40L preincubation. The percent reduction, was much less pronounced compared with suppression of IL-12 (mean ± SD, 23 ± 10.3%) in cells positive for IL-6 by preincubation with sCD40L (n = 4).



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  		FIGURE 1. Effect of preincubation with sCD40L on intracellular IL-12 (p40/p70) production by human monocytes. A, Preincubation (pre) of human monocytes was performed for 16 h using 2 µg/ml sCD40L. Monocytes were washed after the preincubation and stimulated with 300 U/ml IFN-{gamma} and 50 ng/ml LPS (or 2 µg/ml sCD40L) for 24 h. The percentages of monocytes positive for IL-12 (p40/p70), as detected by flow cytometric measurement, are shown. Results are given as the mean ± SD of five independent experiments. B, In a second series of experiments sCD40L was used as the second signal in the stimulation procedure instead of LPS, resulting in >80% of the cells being positive for IL-12p40/p70. Soluble CD40L was used during preincubation, followed by a washing step and subsequent stimulation with IFN-{gamma} and sCD40L, resulting in a complete suppression of IL-12 production. IFN-{gamma} alone did not lead to any significant production of IL-12. A representative experiment of 12 is depicted.

 


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  		FIGURE 2. Kinetics of IL-12 suppression by sCD40L. Human monocytes were stimulated with IFN-{gamma} (300 U/ml) and LPS (50 ng/ml) for 24 h. sCD40L (2 µg/ml) was added at different time intervals before the stimulation procedure. The binding of PE-labeled mouse anti-human IL-12 (p40/p70) is shown (vertical; horizontal, forward scatter). Quadrants were set according to the isotype-matched controls. The percentages of IL-12-positive cells are given. One representative of two independent experiments is depicted.

 


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  		FIGURE 3. Effect of sCD40L preincubation on expression of cell surface molecules. The binding of PE-labeled CD80/CD86 to monocytes is shown. An overlay histogram is given to compare membrane expression of costimulatory molecules on unstimulated cells (filled histogram), monocytes stimulated with IFN-{gamma}/LPS (open histogram, dotted line), and IFN-{gamma}/LPS-stimulated monocytes preincubated with sCD40L for 16 h (open histogram, solid line). One representative experiment of a total of three is depicted.

 
Suppression of IL-12 by preincubation with ligands of the TNFR family is not due to IL-4, IL-10, PGE2, increased apoptosis, or receptor down-modulation

We used neutralizing IL-10, IL-4 Abs, and indomethacin to exclude that the effect was due to endogenous production of IL-10, IL-4, or PGE2, which all have been shown to be potent suppressors of IL-12 under certain conditions. None of these mediators was responsible for the IL-12 suppression (Fig. 4GoA). The effects of sCD40L, sRANKL, or TNF-{alpha} on the inhibition of IL-12 production was also reproducible when we used PBMC instead of isolated monocyte fractions, which excludes T cell-derived mediators to play a pivotal regulatory role in this experimental setting.



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  		FIGURE 4. Effect of blocking IL-10, IL-4, or PGE2 synthesis on sCD40L-induced IL-12 suppression and receptor expression upon sCD40L preincubation. A, Immunofluorescence was performed with human monocytes stimulated with IFN-{gamma} and LPS (all except the medium control). Preincubation was performed with 2 µg/ml sCD40L alone or in the presence of neutralizing anti IL-10, neutralizing anti-IL-4 Ab, or indomethacin. The binding of PE-labeled mouse anti-human IL-12 (p40/p70) is shown. Quadrants were set according to the isotype-matched controls. The percentages of cells in the corresponding quadrants are given. B, Receptor expression on unstimulated monocytes (filled histogram) is compared with the expression after 16-h preincubation with sCD40L. FITC-labeled CD40/IFN-{gamma}R Abs were used. One representative experiment of a total of three is depicted.

 
Monocytes did not become unresponsive to the priming/challenging signal because of receptor down-regulation as CD40 and IFN-{gamma}R (and CD14) (37) (data not shown) expression were not reduced by preincubation with the second signal (Fig. 4GoB). Moreover, the suppression of IL-12 production was not due to increased numbers of monocytes undergoing apoptosis upon preincubation followed by stimulation; staining with annexin V and propidium iodide revealed no significant difference between monocytes preincubated with CD40L and stimulated with IFN-{gamma}/LPS and monocytes stimulated with IFN-{gamma}/LPS without preincubation (data not shown).

Suppression of IL-12 affects the mRNA level

Using real-time fluorescence PCR, a quantitative analysis of IL-12p40 and IL-12p35 suppression was performed, showing that mRNA accumulation of both subunits was significantly reduced by sCD40L (Fig. 5Go, p35 not shown). A similar reduction in IL-12p40 mRNA accumulation was observed using sRANKL for preincubation. Thus, the suppression of IL-12 production seems to be regulated at the mRNA level.



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  		FIGURE 5. IL-12p40 mRNA expression in human monocytes upon preincubation with sCD40L. RT-PCR was performed with IFN-{gamma}/LPS-stimulated monocytes (or unstimulated monocytes) preincubated with sCD40L alone or in the presence of the MEK1/2-specific inhibitor PD98056. A real-time PCR was performed for IL-12p40 and {beta}-actin using the dsDNA binding dye SYBR Green. The results are shown as the intensity of the fluorescence signal (F) vs cycle number plot. The melting peak data clearly show the amplification of one specific product with a melting peak of 88.2°C for IL-12p40 and 90°C for {beta}-actin. The relative amounts of IL-12p40 signal (as internal standard a serial dilution of cDNA derived from IFN-{gamma}/LPS-stimulated monocytes amplified along with the samples was used) were determined and referred to the {beta}-actin amounts. The results of four independent experiments are summarized in the diagram.

 
Evidence for involvement of the p44/42 MAPK pathway in suppression of IL-12

To analyze whether CD40L-induced down-regulation of IL-12 p40 mRNA is controlled by ERK signaling we performed experiments in the presence of specific inhibitors of the MAPK pathway. No selective inhibitor of ERK is available at present. However, the MEK inhibitor PD98059, which primarily inhibits MEK1/2 activation by blockade of the access of activating enzymes (37), or U0126, which is able to inhibit the activated, phosphorylated form of MEK1/2 (38) can be used to inhibit MEK-dependent ERK activation. Hence, we examined the production of IL-12 upon CD40L preincubation in the presence of PD98059, followed, after a washing step, by stimulation with IFN-{gamma}/LPS. Inhibition of MEK1/2 by PD98059 led to an increase in IL-12p40 mRNA levels (Fig. 5Go). Similar results as with PD98059 were obtained with U0126.

Moreover, strong increases in intracellular IL-12 protein were detected in monocytes treated with sCD40L or sRANKL and MEK inhibitors compared with monocytes that were preincubated with these TNFR family ligands in the absence of MEK inhibitors (Fig. 6Go). SB202474 was used as a negative control for the MAPK inhibition studies and did not show an increase in IL-12 production if given during preincubation with sCD40L.



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  		FIGURE 6. Effect of MEK1/2 inhibition by PD98059 on sCD40L/sRANKL-induced IL-12 suppression. Immunofluorescence was performed with human monocytes (3 x 105 cells/well) stimulated with IFN-{gamma} (300 U/ml) and LPS (50 ng/ml). Preincubation, followed by a change of culture medium, was performed with sCD40L/sRANKL alone or sCD40L/sRANKL in the presence of PD98059 (60 µM) for 16 h. The percentages of monocytes positive for IL-12 (p40/p70), as detected by flow cytometry, were determined. A, A stimulation index was calculated using LPS/IFN-{gamma}-stimulated cells without pretreatment as a positive control (100%). Results are given as the mean ± SD of six independent experiments. B, The binding of PE-labeled mouse anti-human IL-12 (p40/p70) is shown for a representative experiment (vertical; horizontal, forward scatter). Quadrants were set according to the isotype-matched controls. The percentages of cells in the corresponding quadrants are given.

 
Western blot analysis showed that stimulation with sCD40L during the preincubation period induces phosphorylation of ERK1/2 within 15 min (Fig. 7GoA). When we compared monocytes that had either been treated with sCD40L and subsequently with IFN-{gamma} to monocytes that had been treated with IFN-{gamma} only we did not observe differences in Stat1 phosphorylation (Fig. 7GoB). Furthermore, we analyzed signal transduction events in monocytes stimulated with the priming and second signals with or without a preceding contact to sCD40L. In these monocytes we could not detect any differences in p38 phosphorylation and also could not detect enhanced p44/42 phosphorylation in those cells that underwent sCD40L preincubation.



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  		FIGURE 7. ERK1/2 and Stat1 activation in differentially stimulated monocytes. A, Monocytes were stimulated with sCD40L (2 µg/ml). After 15 min, cell lysates were harvested for Western blot analysis of phosphorylated p44/42 MAPK (Thr202/Tyr204). In independent experiments with three different donors, ERK1/2 was detected upon sCD40L stimulation. B, Monocytes were stimulated with IFN-{gamma} only (first lane) or in addition were preincubated with sCD40L. Cell lysates for Western blotting (phosphorylated Stat1 (Tyr701)) were obtained 15 min after IFN-{gamma} stimulation. Five donors were tested in independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-12 is considered to play a pivotal role in the development of Th1 cells. Therefore, understanding the mechanisms of IL-12 production is important for the analysis of immune responses and may provide a means to manipulate Th1 and Th2 responses.

In the present paper, we provide evidence that the sequence of stimuli encountered by human monocytes has an important regulatory impact on IL-12 synthesis. Our results highlight the emerging view that the timing and level of costimulation, rather than simply its presence or absence, are critical determinants for the outcome of the immune responses.

Induction of IL-12 production by human APC is dependent on stimulation with two signals, a priming one (i.e., IFN-{gamma} or GM-CSF), followed by a second, challenging signal (i.e., LPS). In this study, we demonstrate that unstimulated, unprimed human monocytes fail to produce high amounts of IL-12 if the second signal (LPS, TNF-{alpha}, or sCD40L) precedes the priming signal (IFN-{gamma} or GM-CSF). Previous work from our group and others pointed to a down-regulation of IL-12 by preincubation with LPS (34, 39) and TNF-{alpha} (36, 40), but this is the first time that physiologic stimuli important to T cell-APC interactions in the in vivo situation are shown to exert a down-regulatory effect on human monocytes.

The production of bioactive IL-12 is well documented to be regulated by various cytokines. The most potent IL-12 counter-regulatory mediators have been shown to be IL-10, IL-4, and PGE2 (26, 41, 42), all of which could be excluded as being causal for the phenomenon of IL-12 suppression observed in this study. In addition, endogenous production of counter-regulatory lymphokines could be ruled out as being responsible for the phenomenon, as the same degree of IL-12 suppression was observable using either pure monocytes or PBMC preincubated with CD40L. In conclusion, the down-regulation of IL-12 production is not due to endogenous production of known IL-12 antagonists during preincubation. Moreover, we could show that it is not due to receptor down-regulation (shown for CD40, IFN-{gamma}R, and CD14), thus ruling out that monocytes are unable to respond to the following stimulation procedure.

It has been shown that repeated and strong stimulation can drive cells into apoptosis. In our experiments we ruled out that the reduced IL-12 signal was due to an increased number of monocytes undergoing apoptosis upon preincubation with CD40L, followed by stimulation with IFN-{gamma}/LPS compared with cells stimulated with IFN-{gamma}/LPS without any preincubation. This is supported by data from the literature pointing to an anti-apoptotic action of CD40L on monocytes and DC (43, 44, 45, 46, 47).

The observed down-regulation seems to affect IL-12-inducing pathways in particular, as the release of IL-6 is only moderately down-regulated by preincubation with the second signal. Of note, in many other reports on the down-regulation of IL-12, IL-6 was used as a control monokine that was not or was only moderately down-modulated (26, 27, 48, 49). Interestingly, van der Pouw Kraan et al. (26) could show that the moderate down-regulation of IL-6 compared with IL-12 by PGE2 was due to endogenous production of IL-10, which was not responsible for IL-12 reduction. Moreover, a general state of hyporesponsiveness of monocytes preincubated with sCD40L could also be ruled out, as the expression of CD80 and CD86 was not reduced under the same experimental setting.

A number of IL-12 down-regulatory agents have been described that may act via different pathways. Some of these agents have been shown to increase intracellular cAMP levels (23, 26, 27, 49). However, other IL-12 inhibitors, such as iC3b (25), ligation of phagocytic receptors (50), measles virus (24), 1,25-dihydroxyvitamin D3 (21), and IL-10, are not thought to suppress IL-12 via the induction of cAMP.

In monocytic cells priming signals (i.e., IFN-{gamma}) enhance IL-12 production probably by rendering second-signal-induced transcription of IL-12 genes possible (18). Preincubation with a second signal initiates a signal transduction event that obviously prevents monocyte priming for IL-12 production. According to our data, responsiveness to IFN-{gamma} is still present, as Stat1 phosphorylation is not changed. Therefore, changes upon preincubation may have occurred further downstream, probably affecting protein-DNA interaction in the promoter region. At this site, different inhibitor-induced pathways may lead to a common downstream event. Other agents, such as C5a, which has previously been described by us to inhibit IL-12 production if given after the priming signal (28), may render primed monocytes insensitive to subsequent stimulation with the second signal. Taken together and in view of the data presented here, it becomes evident that IL-12 suppression is regulated via different intracellular pathways, all of which are not yet completely understood. Negative regulatory sites within the IL-12 promoter have not yet been fully identified. Plevy et al. (17) suggested that liver-enriched transcriptional inhibitory protein (a C/EBP{beta} isoform) may act as a negative regulatory transcription factor. As for positive regulatory sites within the IL-12 p40 promoter, an Ets site (51), an NF-{kappa}B half-site (16), and a C/EBP element (17) have been described. Further studies in this field are clearly needed.

Several members of the TNFR superfamily, such as CD40 and RANK, play critical roles in the initiation and regulation of the immune response (1). The CD40R plays a dominant role in enhancing APC functions (52); however, the signaling pathways activated through CD40 have not been clearly elucidated. It is of interest in relation to the data presented in this paper that a CD40-dependent phosphorylation/activation of ERK1/2 in human APC has been reported recently in independent studies (32, 53, 54). However, the signaling pathway coupling CD40 to ERK activation has remained unknown, but TNFR-associated factor 6 may play an important role (55). Pearson et al. (53) have shown that engagement of CD40 in monocytes led to a rapid and transient activation of ERK1 and 2 and to low levels of c-Jun amino-terminal kinase activation. No CD40-dependent activation of p38 MAPK was found. Vidalain et al. (32) showed that for human monocyte-derived dendritic cells CD40 molecules associate within membrane rafts, and these microdomains provide a platform for CD40 signaling, i.e., TNFR-associated factors 2 and 3 recruitment to the CD40 cytoplasmic tail and activation of the Lyn Scr family kinase. They provide evidence that CD40-mediated Src family kinase activation initiates a pathway that implicates ERK activation. The IL-12 promoter element that confers its MAPK dependency is not identified yet.

The data presented here indicate that IL-12 production is negatively regulated by CD40-activated ERK. In contrast, it is clear that CD40 also activates signaling pathways that stimulate IL-12 production (31, 32, 33). Apparently, both stimulatory and inhibitory pathways can be activated by the same stimulus (sCD40L) depending on the cellular and temporal context. Depending on the timing the physiologic self-signal CD40L can regulate IL-12 production by activating MAPK cascades. Regulating the MAPK pathways may turn out to be a promising target for immune modulation. In that light Feng et al. (29) demonstrated in a murine system that pathogens may act to evade the host immune response by subverting host MAPK regulation of the macrophage effector response, and that Leishmania may suppress resistance to infection by switching on ERK MAPK-mediated negative regulation of IL-12 production and hence preventing generation of a protective Th1 immune response.

Further studies are clearly needed to elucidate whether modulation of IL-12-regulating signal transduction pathways may be helpful in disease states where the inappropriate production of IL-12 may contribute to immune deviation, such as autoimmune (56) or allergic diseases.


   Acknowledgments
 
We thank Tina Tanneberg for excellent technical assistance.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 566, A6. Back

2 Address correspondence and reprint requests to Dr. Miriam Wittmann, Department of Dermatology and Allergology, Hannover Medical University, Ricklinger Strasse 5, D-30449 Hannover, Germany. E-mail address: miriamwittmann{at}web.de Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; ERK, extracellular signal-related kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, ERK-specific MAPK kinase; sCD40L, soluble CD40L; RT, reverse transcription; TRANCE, TNF-related activation-induced cytokine; RANK, receptor activator of NF-{kappa}B; RANKL, RANK ligand. Back

Received for publication May 11, 2001. Accepted for publication February 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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