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Originally published online as doi:10.1189/jlb.1006633 on March 9, 2007

Published online before print March 9, 2007
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(Journal of Leukocyte Biology. 2007;81:1562-1567.)
© 2007 by Society for Leukocyte Biology

Reciprocal effects of IFN-ß and IL-12 on STAT4 activation and cytokine induction in T cells

Angela J. Fahey*, R. Adrian Robins{dagger} and Cris S. Constantinescu*,1

Divisions of
* Clinical Neurology and
{dagger} Molecular and Clinical Immunology, University of Nottingham, Nottingham, United Kingdom

1 Correspondence: Division of Clinical Neurology, Queens Medical Centre, Medical School, University of Nottingham, Nottingham NG7 2UH, UK. E-mail: cris.constantinescu{at}nottingham.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-12 is an immunoregulatory cytokine, which promotes Th1 cell differentiation and is a major inducer of IFN-{gamma}. IFN-ß, a Type I IFN used in the treatment of multiple sclerosis, has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10, a major suppressor of Th1 cytokines. The beneficial immunomodulatory effects of IFN-ß may in part be a result of its ability to suppress IL-12. However, IL-12 and IFN-ß signal via the STAT4 pathway. Our aim was to investigate the relationship between IL-12 and IFN-ß by observing the effect of prior exposure to IL-12 or IFN-ß on the ability of T cells to subsequently respond to the other cytokine. We report that IFN-ß increases IL-12-induced STAT4 phosphorylation and up-regulates IL-12 receptor ß1 and ß2 expression. However, despite this up-regulation, IFN-ß suppressed IL-12-induced IFN-{gamma} expression. Our results suggest that this may be a result of the parallel induction of IL-10 by IFN-ß.

Key Words: human • signal transduction • IFN-gamma • interleukin-10


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-12 plays a central role in the regulation of cellular immune responses, mainly as a result of its ability to induce the production of IFN-{gamma} by effector cells of the innate and adaptive immune systems [1 ]. Previous studies have indicated that although IL-12 exerts effects that are independent of IFN-{gamma}, induction of Th1 cells is reduced when IFN-{gamma} is blocked [2 ]. IL-12 acts via the STAT4 signaling pathway. Signal transduction through the IL-12 receptor (IL-12R) induces tyrosine phosphorylation of the Janus family kinases JAK2 and TYK2, which in turn phosphorylate STAT4 [3 ]. Phosphorylated STAT4 (pSTAT4) dimerizes, translocates to the nucleus, and instigates DNA transcription [4 ]. IL-12 is critical in the development of Th1 differentiation [1 ], is protective in a variety of intracellular infections, and has shown potent antitumor activity [5 , 6 ]. However, IL-12 also has a clear potential to cause injury to the host and is thought to contribute to the pathogenesis of immune-mediated, inflammatory disorders including multiple sclerosis (MS) and rheumatoid arthritis [7 ].

The Type I interferon, IFN-ß, is the best-characterized and most-used disease-modifying treatment for MS. It has been demonstrated that IFN-ß significantly increases the expression of the anti-inflammatory cytokine IL-10 [8 ], a major suppressor of Th1 cytokines [9 , 10 ]. Like IL-12, IFN-ß also acts via the STAT4 pathway.

The beneficial immunomodulatory effects of IFN-ß may in part be a result of its ability to suppress IL-12 [11 ]; however, IFN-ß has also been shown to up-regulate IL-12Rß2 chain expression [12 ]. Immunoregulatory cytokine networks are rarely simple, and the relationship between IL-12 and Type I IFNs is no exception. As IL-12 and IFN-ß signal via the STAT4 pathway, we wanted to investigate the effects of one cytokine on STAT4 activation by the other and the downstream influences on important biological effects of these molecules such as cytokine induction. We observe the effect of prior exposure to IL-12 or IFN-ß on the ability of T cells to subsequently respond to the other cytokine.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The Nottingham Research Ethics Committee (UK) approved this study.

Cytokines and antibodies
The following reagents were obtained: recombinant human IL-12 (PeproTech EC, London, UK), IFN-ß (Rebif®, a gift of Dr. Paul Byrne, Serono, London, UK), and polyclonal rabbit anti-pSTAT4 antibody (Zymed Laboratories, Inc., San Francisco, CA, USA). Antihuman CD3-PE mAb, antihuman IFN-{alpha} receptor-1 (IFNAR-1) mAb, antihuman IFN-{gamma}-FITC mAb, antihuman IL-10-IgG2a mAb (unlabeled), antihuman IL-12Rß1-PE mAb, and mouse IgG isotype control were purchased from R&D Systems (Oxford, UK). Purified rat antihuman IL-12Rß2 mAb was purchased from BD PharMingen (San Diego, CA, USA). Antihuman IL-10-PE mAb, goat antimouse-FITC secondary antibody, goat antirabbit-PE secondary antibody, and goat antirat-PE secondary antibody were purchased from Caltag Laboratories (S. San Francisco, CA, USA). IFN-{gamma} and IL-10 solid-phase sandwich ELISAs were purchased from PharMingen (BD OptEIATM BD Biosciences, Oxford, UK). CD4+ and CD8+ T cell-negative selection kits were purchased from StemCell Technologies Inc. (London, UK).

Cell preparation
PBMC from healthy donors were isolated by standard gradient centrifugation with Histopaque 1077 (Sigma-Aldrich, Dorset, UK). The mononuclear cells were prepared at 1 x 106 cells/ml in media consisting of RPMI 1640, 2 mM glutamine, 20 mM Hepes, 0.1 mg/ml penicillin and streptomycin, and 10% FCS (Sigma-Aldrich). The cells were cocultured with 10 µg/ml PHA (Sigma-Aldrich) at 37°C and 5% CO2 for 72 h. Following PHA-induced proliferation, the cells were washed with media and stimulated with 100 U/ml IL-2 (R&D Systems, Minneapolis, MN, USA) at 37°C and 5% CO2 for a further 24 h. The cells were then allowed to rest for 24 h in serum-free media under the same conditions.

Cell stimulation
PHA/IL-2-induced T cell blasts (1x106cells/ml) were left untreated or treated with 10 ng/ml IFN-ß or 0.1 µg/ml IL-12 at 37°C for 30 min. Subsequently, the cells were left unstimulated or incubated with 10 ng/ml IFN-ß or 0.1 µg/ml IL-12 at 37°C for a further 30 min. The method used was refined so that the optimum concentrations of cytokines were used. Varying concentrations of IFN-ß and IL-12 were analyzed for their effect on STAT4 phosphorylation; the concentrations used in this experiment were those that produced the peak pSTAT4 value. The effect of duration of stimulation on STAT4 phosphorylation was also observed. Optimal stimulation was achieved after 30 min.

PBMC-derived CD3+ cells were isolated by FACS sorting. PBMC were washed by centrifugation in 2% FCS RPMI, the supernatant was poured off, and the cells were resuspended in the residue, to which 10 µl antihuman CD3-PE-labeled mAb was added and incubated on ice for 30 min. The cells were washed again by centrifugation in 2% FCS RPMI. Cells were sorted in 1 ml 10% FCS RPMI and left overnight in fresh media until stimulation. CD4+ T cells and CD8+ T cell populations were separated using an immunomagnetic cell selection procedure using EasySep® mAb, magnetic nanoparticles, and magnet following the manufacturer’s protocol (StemCell Technologies Inc.). The sorted cells were also left overnight in fresh media until stimulation.

Intracellular staining
Intracellular staining for STAT4, IFN-{gamma}, and IL-10 was done as described previously [13 ]. Briefly, the cells were fixed in ice-cold 70% ethanol and incubated on ice for 20 min. The cells were then washed by centrifugation at 300 g for 5 min. The supernatant was poured off, and the cells were resuspended in the residue, to which 0.5 µg primary STAT4 or pSTAT4 antibody or 10 µl antihuman IFN-{gamma}-FITC mAb or antihuman IL-10-PE mAb was added and incubated for 30 min. Following incubation, the cells were washed by centrifugation with saponin buffer. The cells incubated with STAT4 or pSTAT4 primary antibodies were incubated with the secondary antibody, 1 µg PE-conjugated goat antirabbit IgG for 30 min. All cells were washed with saponin buffer before resuspension in 0.5% formaldehyde for flow cytometry.

Surface staining
Following incubation with IFN-ß or IL-12, the cells were washed by centrifugation in 2% FCS RPMI, the supernatant was poured off, and the cells were resuspended in the residue, to which 10 µl IFNAR-1, IL-12Rß1-PE-labeled, or IL-12ß2 antibodies were added and incubated on ice for 30 min. The cells were washed again by centrifugation in 2% FCS RPMI. Those cells incubated with nonconjugated, primary antibodies (IFNAR and IL-12Rß2) were incubated with the secondary antibody, 5 µl goat antimouse FITC and goat anti-rat-PE, respectively, and incubated for a further 30 min on ice. All cells were washed twice in 1 ml 2% FCS RPMI before resuspension in 500 µl 0.5% formaldehyde for flow cytometry.

Cytokine assay using ELISA
PHA/IL-2-induced T cell blasts (1x106cells/ml) were left untreated or pretreated with 10 ng/ml IFN-ß or 0.1 µg/ml IL-12 at 37°C for 18 h. Subsequently, the cells were left unstimulated or incubated with 10 ng/ml IFN-ß or 0.1 µg/ml IL-12 at 37°C for a further 18 h. The cell supernatants were removed for cytokine assay. IFN-{gamma} and IL-10 levels were measured using a solid-phase sandwich ELISA following the manufacturer’s instructions.

Small interfering (si)RNA transfection
Approximately 24 h before transfection, PHA/IL-2 T cell blasts were plated in normal growth medium (10% FCS RPMI) so that they would be 50–70% confluent after 24 h. Transfection was performed using the transfection reagent siPORT Amine (Ambion, Inc., Austin, TX, USA) following the manufacturer’s instructions. siRNA to target STAT4 (Ambion, siRNA ID 4595) and Silencer® negative-control siRNA (Ambion) were transfected. Initially, the cells were cultured for 6 h with the siRNA in 200 µl media. Following this incubation, 1 ml fresh medium (10% FCS RPMI) was added to each well. The cells were cultured for a further 24 h to maximize cell growth and prevent potential cytotoxicity. The cells were then harvested, and the level of siRNA-mediated STAT4 silencing was measured using flow cytometry. Transfected and nontransfected cells were stimulated with 10 ng/ml IFN-ß; IL-10 production was also measured by flow cytometry.

Neutralization of human IL-10 bioactivity
PHA/IL-2-induced T cell blasts (1x106 cells/ml) were left untreated or pretreated with 0.5 µg/ml mouse antihuman IL-10 antibody to neutralize its bioactivity or 0.5 µg/ml mouse IgG isotype control antibody as a negative control. The cells were left untreated or pretreated with 10 ng/ml IFN-ß for 18 h and then were subsequently incubated with 0.1 µg/ml IL-12 at 37°C for a further 18 h. Following incubation, the cells were stained intracellularly for IFN-{gamma} and analyzed using flow cytometry.

Measurements
The PHA/IL-2 blasts were evaluated following antibody staining on an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, USA), and the results were analyzed using the computer software WINMDI 2.8. Each experiment was repeated five times, using five independent donors (see Table 1 ). IFN-{gamma} and IL-10 levels were also measured using a solid-phase sandwich ELISA. Statistical analysis was performed using the Friedman nonparametric test for multiple related groups. To compare between paired groups, the Wilcoxon test was performed.


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  		Table 1. IFNAR, IL-12Rß1, and IL-12Rß2 Expression in Human T Cell Blasts after Stimulation with IFN-ß or IL-12

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of STAT4 by IL-12 and IFN-ß
As shown in Figure 1 , we first confirmed previous results showing that IL-12 and IFN-ß increase pSTAT4. Prior incubation with IFN-ß, followed by stimulation with IL-12, resulted in an increase in pSTAT4 generation compared with cells incubated with IL-12 alone (Fig. 1A) . Prior exposure to IL-12 followed by stimulation with IFN-ß resulted in similar pSTAT4 generation when compared with cells incubated with IFN-ß alone (Fig. 1B) .


Figure 1
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  		Figure 1. pSTAT4 in human PBMC-derived PHA/IL-2 T cells. Cells were left unstimulated or incubated with 0.1 µg/ml IL-12 and 10 ng/ml IFN-ß for 30 min at 37°C, pretreated with 10 ng/ml IFN-ß, followed by 0.1 µg/ml IL-12 (A), or pretreated with 0.1 µg/ml IL-12, followed by 10 ng/ml IFN-ß (B). IL-12 and IFN-ß increase pSTAT4 generation compared with unstimulated cells (P<0.05). Pretreatment with IFN-ß and then IL-12 resulted in an increase in pSTAT4 generation (P<0.05; A), and IFN-ß STAT4 activation was unaffected by prior exposure to IL-12 (B).

 
The above experiments were also performed on CD3+ cells isolated by FACS sorting and CD4+ and CD8+ T cell populations separated using an immunomagnetic cell-selection procedure. For the CD3+ cells, the same results, as observed with the PHA/IL-2 cell blasts, were obtained. Incubation with IFN-ß and then IL-12 resulted in an increase in pSTAT4 generation, and prior treatment with IL-12 followed by IFN-ß resulted in similar pSTAT4 generation when compared with IFN-ß alone (data not shown). This was also true for the CD4+ and CD8+ T cell populations (Fig. 2A and 2B ).


Figure 2
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  		Figure 2. pSTAT4 in human PBMC-derived CD4+ or CD8+ T cells. PBMC were separated into CD4+ (A) or CD8+ (B) T cell populations. Cells were left unstimulated, incubated with 0.1 µg/ml IL-12 or 10 ng/ml IFN-ß for 30 min at 37°C, or pretreated with 10 ng/ml IFN-ß, followed by 0.1 µg/ml IL-12. IL-12 and IFN-ß increase pSTAT4 generation compared with unstimulated cells. Pretreatment with IFN-ß and then IL-12 resulted in an increase in pSTAT4 generation.

 
IL-12R and IFNR expression
We next investigated upstream of STAT4, at the receptor level, to determine whether the effects observed on STAT4 were a result of modulation of the receptors for the cytokines studied.

Our results show that IFN-ß increases subunits ß1 and ß2 of the IL-12R, in part, consistent with previous studies showing IFN-ß to increase IL-12Rß2 [14 ] (IL-12Rß1 expression was not reported in that study). No change was observed in IFN-ßR expression by IL-12 exposure (Table 1 ).

Induction of IFN-{gamma} and IL-10 by IL-12 and IFN-ß
We next determined whether the reciprocal effects on receptor and STAT4 signaling translate into influences on key biological effects of IFN-ß and IL-12.

We show that IFN-ß induces IL-10 production in human T cells, confirming previous results [8 , 15 ]. Incubation with IL-12 prior to IFN-ß stimulation resulted in similar IL-10 generation when compared with cells incubated with IFN-ß alone (Fig. 3A ). These results were confirmed using ELISA (Fig. 3C) . As expected, we also show increased production of IFN-{gamma} by IL-12 in human T cells. However, pretreatment of these cells with IFN-ß reduced the induction of IFN-{gamma} by IL-12 (Fig. 3B) . Again, these results were confirmed using ELISA (Fig. 3C) .


Figure 3
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  		Figure 3. IFN-ß (10 ng/ml) induces IL-10 production in human T cells (shaded curve, A; P<0.05). IL-10 production remains similar after prior exposure of the cells to IL-12 (A; solid, unshaded curve, labeled, IL-12 pretreatment followed by IFN-ß stimulation). IL-12 (0.1 µg/ml) induces IFN-{gamma} production in human T cells (shaded curve B; P<0.05). Pretreatment with IFN-ß (10 ng/ml) reduces this induction (B; P<0.05; solid, unshaded curve, labeled, IFN-ß pretreatment followed by IL-12 stimulation). Dotted line represents unstimulated cells (US). IFN-{gamma} and IL-10 levels were also measured using a solid-phase sandwich ELISA (C). Bars denote the median value, and error bars represent range. IL-12 (0.1 µg/ml) induces IFN-{gamma} production (P<0.05); this was reduced by prior treatment with IFN-ß (P<0.05). For the ELISA, the duration of exposure of the cells to IFN-ß and IL-12 was 18 h.

 
siRNA-mediated gene silencing in T cell blasts
We used RNA interference [13 , 16 ] with a STAT4 siRNA to determine whether the effects shown above were directly a result of the known IL-12 and IFN-ß effects on STAT4 activation. Ambion Silencer® negative-control siRNA was used to demonstrate that the transfection itself did not induce nonspecific effects on gene expression. The negative-control siRNA are comprised of a scrambled sequence, which has no significant homology to any known gene sequence.

Transfection with the siRNA to target STAT4 resulted in 90% reduction in STAT4 expression as measured by flow cytometry (Fig. 4 ). This reduction was not observed when just the transfection agent or the scrambled siRNA was added.


Figure 4
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  		Figure 4. siRNA transfection, which with the siRNA to target STAT4, resulted in 90% reduction in STAT4 expression as measured by flow cytometry (A). No reduction was seen with scrambled siRNA or transfection agent only (data not shown). IFN-ß induces IL-10 production in nontransfected T cell blasts (B). In STAT4, siRNA-transfected cells, IFN-ß-induced IL-10 was reduced (unshaded curve). T cells were left untreated or pretreated with 0.5 µg/ml neutralizing antihuman IL-10 antibody (Ab) or 0.5 µg/ml mouse IgG2a (C). Cells were left untreated or pretreated with 10 ng/ml IFN-ß and then incubated with 0.1 µg/ml IL-12. The cells were then stained for IFN-{gamma} and analyzed using flow cytometry. IL-12 induced IFN-{gamma} production (C). Pretreatment with IFN-ß in the absence of IL-10 antibody reduces IFN-{gamma} induction by IL-12. In the presence of IL-10 antibody but not the control antibody, the observed suppression of IFN-{gamma} by IFN-ß was abrogated. Unstimulated, Transfected, unstimulated cells. A similar curve was obtained from nontransfected, unstimulated cells. Representative results of four independent experiments.

 
As already observed in this study, IFN-ß induced IL-10 production in nontransfected T cells blasts. However, this induction was reduced when STAT4 gene expression was silenced by siRNA (see Fig. 4B ), thus suggesting that the STAT4 signaling pathway is in some way involved in the ability of IFN-ß to induce IL-10 production.

Neutralization of IL-10 bioactivity
In addition to enhancing the level of STAT4 activation by IL-12, IFN-ß up-regulated its receptor. Thus, one would expect an enhancing effect of IFN-ß on biological effects of IL-12, including IFN-{gamma} induction. However IFN-ß suppressed IL-12-induced IFN-{gamma} expression. As shown, IFN-ß induces IL-10 production, and IL-10 and IFN-{gamma} antagonize one another [17 , 18 ]; therefore, we postulated that exposure to IFN-ß induces IL-10, which in turn inhibits IFN-{gamma} production. To investigate the role of IL-10 in the IFN-ß-mediated suppression of IL-12-induced IFN-{gamma}, we used a neutralizing anti-IL-10 antibody. In the presence of this IL-10 antibody, the suppression of IL-12-induced IFN-{gamma} by IFN-ß was abrogated (see Fig. 4C ), and therefore, no significant difference in IFN-{gamma} production was observed between IL-12-induced cells and IFN-ß; then IL-12 stimulated cells in the presence of IL-10 antibody (P=0.68). In the presence of the isotype-matched control antibody, IFN-ß suppressed IL-12-induced IFN-{gamma} production (Fig. 4C) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-12 is the major cytokine that induces the development of Th1 cells. Many effects associated with IL-12 are attributable to the induction of IFN-{gamma} [19 ]. IFN-ß also has a variety of immunoregulatory effects [20 ] and has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10 [8 , 15 ]. IFN-ß has the ability to inhibit IL-12 induction and block its downstream effects [20 ]. Conversely, it increases the induction of the IL-12Rß2 [12 ].

In MS, IFN-ß dampens the Th1 response [8 ], and IL-12 promotes Th1 cell differentiation [19 , 21 ]. However, IL-12 and IFN-ß signal via STAT4. Our aim, therefore, was to investigate the effects of one cytokine on STAT4 activation by the other. Prior exposure to IL-12 did not affect the level of STAT4 activation by IFN-ß. Also, no change was observed upstream at the IFN-Rß level, and there was no effect of IL-12 exposure on IFN-ß-induced IL-10. Previous studies have shown that CD4+ and CD8+ T cells exhibit lineage-specific differences in some aspects of STAT4 signaling [22 , 23 ]. Therefore, we also observed the effects of one cytokine on STAT4 activation by the other on CD4+ and CD8+ cells. The same results as observed with the PHA/IL-2 cell blasts and CD3+ were obtained.

We demonstrated a differential effect of IFN-ß on the ability of T cells to respond to IL-12. We show that prior exposure to IFN-ß enhances the effect of IL-12 on STAT4 activation and up-regulates both IL-12R subunits. However, we also show that IFN-ß does not enhance the ability of IL-12 to induce IFN-{gamma} but instead, reduces the level of IFN-{gamma} induced by IL-12.

Type I IFN-mediated inhibition of IL-12 production was demonstrated, first in murine systems. IFN-{alpha} and IFN-ß inhibited bacterially induced production of IL-12 by murine splenic leukocytes [24 ]. More recently, it has been shown that Type I IFNs can inhibit IL-12 p40 and p70 production by primary human monocytes [11 ]. How Type I IFNs suppress IL-12 production remains unclear; possible mechanisms include inhibition of IFN-{gamma} priming of IL-12 production and the induction of anti-inflammatory cytokines such as IL-10.

We hypothesize that the effect of IFN-ß on IL-10 production is a contributing factor to the suppression of IFN-{gamma} production by IFN-ß. From an immunotherapeutic perspective, an important property of IL-10 is its capacity to inhibit Th1 cells [25 ]. The inhibition of the Th1 cell pathway by IL-10 is mediated by several mechanisms, including inhibition of IL-12 production by APCs and blocking of IFN-{gamma} synthesis by differentiated Th1 cells [26 ].

In this study and previously [15 ], we have shown that IFN-ß induces IL-10 production. Here, we show that by neutralizing IL-10 bioactivity, the observed suppression of IL-12-induced IFN-{gamma} by IFN-ß was abrogated.

In contrast to this study Van Weyenbergh et al. [27 ] concluded that although IFN-ß by itself only induced modest amounts of IFN-{gamma}, IFN-ß was able to synergize with IL-12 for IFN-{gamma} induction. In our study, the cells were pretreated with IFN-ß before being stimulated with IL-12, and in the Van Weyenbergh et al. [27 ] study, the human PBMC were stimulated simultaneously with IFN-ß and IL-12. Under these conditions, there would not be enough time for IL-10 production to be induced first, and therefore, its inhibitory effects may not have been developed, enabling increased IFN-{gamma} production (perhaps through the up-regulation of IL-12R and signaling pathway by IFN-ß).

The IL-12Rß2 chain is the binding and signaling component of the IL-12R, selectively expressed on Th1 cells and crucial for the maintenance of IL-12 responsiveness and Th1 lineage commitment [28 ]. A study by Wandinger et al. [12 ] also showed that induction of the IL-12Rß2 chain by IFN-ß was paralleled by a dose-dependent up-regulation of IL-10 gene expression and that the presence of IFN-ß during antigen-specific T cell activation resulted in a marked suppression of IL-12p40 gene expression and a simultaneous induction of IL-10 transcription. These results suggest that although IFN-ß is observed, as in our study, to up-regulate IL-12R expression, this may not have a proinflammatory, biological consequence, as the concurrent inhibition of IL-12 would result in reduced ligand being available to engage the receptor. The up-regulation of IL-10 would also suppress IL-12 production and thus, reduce the amount of IL-12 even further.

STAT4 signaling is known to promote IFN-{gamma} expression in the human and the mouse, and it has been demonstrated clearly in this study and previously that IFN-ß can activate STAT4 in the human [29 ]. In this study, we have also shown that prior exposure to IFN-ß enhances the effect of IL-12 on STAT4 activation. STAT4 may not be the only pathway to IFN-{gamma} expression in the human. Although stimulation through the TCR for CD4 T cell IFN-{gamma} induction requires STAT4, equivalent stimulation of CD8 T cell IFN-{gamma} responses does not [23 ], suggesting that there are STAT4-independent pathways to IFN-{gamma} expression.

Our siRNA experiments indicate that the STAT4 signaling pathway is in some way involved in the ability of IFN-ß to induce IL-10 production. This finding, also suggested by our previous work [15 ], is interesting. Previously, it has been shown that another Type I IFN, IFN-{alpha}, induces IL-10 via IFN regulatory factor 1 and STAT3 [30 ]. Whether the effect of STAT4 is direct or indirect is subject to our current investigation.

The suppression of IFN-{gamma} by IFN-ß may also be a result of a direct effect on the promoter. However, we think the contribution of this direct effect is likely to be minor, as basal production of IFN-{gamma} in the absence of a stimulus such as IL-12 is much lower. We suggest that the effect of IFN-ß on IL-10 production is an important factor in the suppression of IFN-{gamma} production.

Previous studies have looked at the direct effect of IFN-ß on IL-12 itself [20 , 31 ]. Here, we are more interested in the relationship between IL-12 and IFN-ß with regard to STAT4 activation, and how the ability of T cells to respond to IL-12 by producing IFN-{gamma} is affected by prior exposure to IFN-ß.

In conclusion, despite the up-regulation of IL-12R expression and IL-12 signaling through STAT4 by IFN-ß, the net effect does not appear to be proinflammatory, as a result of the parallel induction of IL-10 by IFN-ß. This is relevant for situations in which these cytokines can coexist, for example, in the treatment of MS. IL-12 is increased [21 ] and presumed detrimental [32 ] in MS, but IFN-ß treatment does not appear to pose the risk of potentiating its proinflammatory effects. It is interesting that the progressive stage of MS is characterized by a loss of the ability of IL-10 to suppress IFN-{gamma} and IL-12 [33 ], and this is the stage in which IFN-ß treatment loses efficacy considerably [34 ].


   ACKNOWLEDGEMENTS
 
This study was supported in part by the Multiple Sclerosis Society of Great Britain and Northern Ireland. We thank Dr. P. Byrne (Serono, London, UK) for the gift of IFN-ß1a (Rebif). We thank Dr. A. M. Grabowska (University of Nottingham) for advice on siRNA.

Received October 13, 2006; revised January 16, 2007; accepted February 2, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Trinchieri, G. (1998) Interleukin-12: a cytokine at the interface of inflammation and immunity Adv. Immunol. 70,83-243[Medline]
  2. Schmitt, E., Hoehn, P., Huels, C., Goedert, S., Palm, N., Rude, E., Germann, T. (1994) T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-{gamma} and is inhibited by transforming growth factor-ß Eur. J. Immunol. 24,793-798[Medline]
  3. Trinchieri, G., Pflanz, S., Kastelein, R. A. (2003) The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses Immunity 19,641-644[CrossRef][Medline]
  4. Bacon, C. M., Petricoin, E. F., III, Ortaldo, J. R., Rees, R. C., Larner, A. C., Johnston, J. A., O’Shea, J. J. (1995) Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes Proc. Natl. Acad. Sci. USA 92,7307-7311[Abstract/Free Full Text]
  5. Mu, J., Zou, J. P., Yamamoto, N., Tsutsui, T., Tai, X. G., Kobayashi, M., Herrmann, S., Fujiwara, H., Hamaoka, T. (1995) Administration of recombinant interleukin 12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes Cancer Res. 55,4404-4408[Abstract/Free Full Text]
  6. Rodolfo, M., Colombo, M. P. (1999) Interleukin-12 as an adjuvant for cancer immunotherapy Methods 19,114-120[CrossRef][Medline]
  7. O’Shea, J. J., Visconti, R. (2000) Type 1 IFNs and regulation of TH1 responses: enigmas both resolved and emerge Nat. Immunol. 1,17-19[Medline]
  8. Rep, M. H., Schrijver, H. M., van Lopik, T., Hintzen, R. Q., Roos, M. T., Ader, H. J., Polman, C. H., van Lier, R. A. (1999) Interferon (IFN)-ß treatment enhances CD95 and interleukin 10 expression but reduces interferon-{gamma} producing T cells in MS patients J. Neuroimmunol. 96,92-100[CrossRef][Medline]
  9. Mosmann, T. R., Moore, K. W. (1991) The role of IL-10 in crossregulation of TH1 and TH2 responses Immunol. Today 12,A49-A53[CrossRef][Medline]
  10. Howard, M., O’Garra, A. (1992) Biological properties of interleukin 10 Immunol. Today 13,198-200[CrossRef][Medline]
  11. Karp, C. L., Biron, C. A., Irani, D. N. (2000) Interferon ß in multiple sclerosis: is IL-12 suppression the key? Immunol. Today 21,24-28[CrossRef][Medline]
  12. Wandinger, K. P., Sturzebecher, C. S., Bielekova, B., Detore, G., Rosenwald, A., Staudt, L. M., McFarland, H. F., Martin, R. (2001) Complex immunomodulatory effects of interferon-ß in multiple sclerosis include the upregulation of T helper 1-associated marker genes Ann. Neurol. 50,349-357[CrossRef][Medline]
  13. Sharp, P. A. (2001) RNA interference—2001 Genes Dev. 15,485-490[Free Full Text]
  14. Rogge, L., Barberis-Maino, L., Biffi, M., Passini, N., Presky, D. H., Gubler, U., Sinigaglia, F. (1997) Selective expression of an interleukin-12 receptor component by human T helper 1 cells J. Exp. Med. 185,825-831[CrossRef]
  15. Fahey, A. J., Robins, R. A., Kindle, K. B., Heery, D. M., Constantinescu, C. S. (2006) Effects of glucocorticoids on STAT4 activation in human T cells are stimulus-dependent J. Leukoc. Biol. 80,133-144[Abstract/Free Full Text]
  16. Hammond, S. M., Caudy, A. A., Hannon, G. J. (2001) Post-transcriptional gene silencing by double-stranded RNA Nat. Rev. Genet. 2,110-119[CrossRef][Medline]
  17. Furlan, R., Bergami, A., Lang, R., Brambilla, E., Franciotta, D., Martinelli, V., Comi, G., Panina, P., Martino, G. (2000) Interferon-ß treatment in multiple sclerosis patients decreases the number of circulating T cells producing interferon-{gamma} and interleukin-4 J. Neuroimmunol. 111,86-92[CrossRef][Medline]
  18. Segal, B. M., Dwyer, B. K., Shevach, E. M. (1998) An interleukin (IL)-10/IL-12 immunoregulatory circuit controls susceptibility to autoimmune disease J. Exp. Med. 187,537-546[Abstract/Free Full Text]
  19. Trinchieri, G. (1995) Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity Annu. Rev. Immunol. 13,251-276[Medline]
  20. Biron, C. A. (2001) Interferons {alpha} and ß as immune regulators—a new look Immunity 14,661-664[CrossRef][Medline]
  21. Drulovic, J., Mostarica-Stojkovic, M., Levic, Z., Mesaros, S., Stojsavljevic, N., Popadic, D., Pravica, V. (1998) Serum interleukin-12 levels in patients with multiple sclerosis Neurosci. Lett. 251,129-132[CrossRef][Medline]
  22. Nguyen, K. B., Watford, W. T., Salomon, R., Hofmann, S. R., Pien, G. C., Morinobu, A., Gadina, M., O’Shea, J. J., Biron, C. A. (2002) Critical role for STAT4 activation by type 1 interferons in the interferon-{gamma} response to viral infection Science 297,2063-2066[Abstract/Free Full Text]
  23. Carter, L. L., Murphy, K. M. (1999) Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon {gamma} production from CD4(+) versus CD8(+) T cells J. Exp. Med. 189,1355-1360[Abstract/Free Full Text]
  24. Cousens, L. P., Orange, J. S., Su, H. C., Biron, C. A. (1997) Interferon-{alpha}/ß inhibition of interleukin 12 and interferon-{gamma} production in vitro and endogenously during viral infection Proc. Natl. Acad. Sci. USA 94,634-639[Abstract/Free Full Text]
  25. Adorini, L. (2003) Cytokine-based immunointervention in the treatment of autoimmune diseases Clin. Exp. Immunol. 132,185-192[CrossRef][Medline]
  26. Moore, K. W., de Waal Malefyt, R., Coffman, R. L., O’Garra, A. (2001) Interleukin-10 and the interleukin-10 receptor Annu. Rev. Immunol. 19,683-765[CrossRef][Medline]
  27. Van Weyenbergh, J., P. Silva, M. P., Bafica, A., Cardoso, S., Wietzerbin, J., Barral-Netto, M. (2001) IFN-ß and TGF-ß differentially regulate IL-12 activity in human peripheral blood mononuclear cells Immunol. Lett. 75,117-122[CrossRef][Medline]
  28. Presky, D. H., Yang, H., Minetti, L. J., Chua, A. O., Nabavi, N., Wu, C. Y., Gately, M. K., Gubler, U. (1996) A functional interleukin 12 receptor complex is composed of two ß-type cytokine receptor subunits Proc. Natl. Acad. Sci. USA 93,14002-14007[Abstract/Free Full Text]
  29. Rogge, L., D’Ambrosio, D., Biffi, M., Penna, G., Minetti, L. J., Presky, D. H., Adorini, L., Sinigaglia, F. (1998) The role of Stat4 in species-specific regulation of Th cell development by type I IFNs J. Immunol. 161,6567-6574[Abstract/Free Full Text]
  30. Ziegler-Heitbrock, L., Lotzerich, M., Schaefer, A., Werner, T., Frankenberger, M., Benkhart, E. (2003) IFN-{alpha} induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3 J. Immunol. 171,285-290[Abstract/Free Full Text]
  31. Van Seventer, J. M., Nagai, T., van Seventer, G. A. (2002) Interferon-ß differentially regulates expression of the IL-12 family members p35, p40, p19 and EBI3 in activated human dendritic cells J. Neuroimmunol. 133,60-71[CrossRef][Medline]
  32. Comabella, M., Balashov, K., Issazadeh, S., Smith, D., Weiner, H. L., Khoury, S. J. (1998) Elevated interleukin-12 in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy J. Clin. Invest. 102,671-678[Medline]
  33. Balashov, K. E., Comabella, M., Ohashi, T., Khoury, S. J., Weiner, H. L. (2000) Defective regulation of IFN{gamma} and IL-12 by endogenous IL-10 in progressive MS Neurology 55,192-198[Abstract/Free Full Text]
  34. Compston, A., Coles, A. (2002) Multiple sclerosis Lancet 359,1221-1231[CrossRef][Medline]




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