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. 2005 Dec;142(3):471–480. doi: 10.1111/j.1365-2249.2005.02933.x

Interferon-α2a is sufficient for promoting dendritic cell immunogenicity

A Tamir *, W J Jordan *, M Ritter *, N Habib ‡, R I Lechler †, G R Foster §, G Lombardi †
PMCID: PMC1809533  PMID: 16297159

Abstract

Type I interferons (IFNs) are widely used therapeutically. IFN-α2a in particular is used as an antiviral agent, but its immunomodulatory properties are poorly understood. Dendritic cells (DCs) are the only antigen-presenting cells able to prime naive T cells and therefore play a crucial role in initiating the adaptive phase of the immune response. We studied the effects of IFN-α2a on DC maturation and its role in determining Th1/Th2 equilibrium. We found that IFN-α2a induced phenotypic maturation of DCs and increased their allostimulatory capacity. When dendritic cells were stimulated simultaneously by CD40 ligation and IFN-α2a, the production of interleukin (IL)-10 and IL-12 was increased. In contrast, lipopolysaccharide (LPS) stimulation in the presence of IFN-α2a mainly induced IL-10 release. The production of IFN-γ and IL-5 by the responder naive T cells was also amplified in response to IFN-α2a-treated DCs. Furthermore, IL-12 production by IFN-α2a-treated DCs was enhanced further in the presence of anti-IL-10 antibody. Different results were obtained when DCs were treated simultaneously with IFN-α2a and other maturation factors, in particular LPS, and then stimulated by CD40 ligation 36 h later. Under these circumstances, IFN-α2a did not modify the DC phenotype, and the production of IL-10/IL-12 and IFN-γ/IL-5 by DCs and by DC-stimulated naive T cells, respectively, was inhibited compared to the effects on DCs treated with maturation factors alone. Altogether, this work suggests that IFN-α2a in isolation is sufficient to promote DC activation, however, other concomitant events, such as exposure to LPS during a bacterial infection, can inhibit its effects. These results clarify some of the in vivo findings obtained with IFN-α2a and have direct implications for the design of IFN-α-based vaccines for immunotherapy.

Introduction

Type I interferons (IFNs) are produced in response to viral [1–3] and non-viral infections [4,5] and may be induced during T cell : dendritic cell (DC) interactions in the absence of infecting agents [6]. Type I IFNs are antiviral cytokines that have a range of immunomodulatory functions [7]. In humans, IFN-α is a multigene family of 13 functional genes, while there is a single gene for IFN-β [8,9]. Although the different IFN-α functional genes have 80–95% homology with each other, and share a common cell surface receptor, they have diverse effects on the cells of the immune response [10,11].

Type I IFNs are used clinically to treat a variety of different disorders. IFN-β reduces the immunopathology of multiple sclerosis (MS) and suppresses interleukin (IL)-12 and augments IL-10 production [12,13]. IFN-α (IFN-α2a, Roferon or IFN-α2b Intron) are used to treat several malignancies [14–16] and chronic viral infections such as hepatitis B, hepatitis C [17,18] and severe acute respiratory syndrome (SARS) [19].

Concurrent with the wide use of type I IFNs in the clinic, the immune functions of these cytokines were investigated in vitro. The effects of IFNs on DC function are contradictory, as some studies showed that type I IFNs promote the differentiation/activation of DCs [20–23] while others reported inhibitory effects [24–26]. In addition, type I IFNs have been shown to act as a maturation factor for DCs and to increase DC maturation [27,28]. Several studies have investigated the direct effect of IFN-α on human CD4+ T cells and showed that IFN-α induced the production of IFN-γ by T cells due to Stat4 phosphorylation [29–32], but others have demonstrated that IFN-α induced the production of both IL-10 and IFN-γ, a cytokine profile which characterizes CD4+ regulatory T cells [33–35]. More recently, we have shown that type I IFNs, in particular IFNα2, induced chemotaxis of T cells and functional changes that resulted in enhanced T cell motility [36].

Similar controversy exists regarding the effects of IFN-α on the production of cytokines by DCs and their effect on T helper cell differentiation. Some studies have shown that DCs generated in the presence of type I IFNs stimulated IFN-γ production by T cells [21, 22, 37], and similar effects on T cell differentiation were obtained when IFN-αβ was added during the maturation of DCs [28,38]. Other groups reported that IFN-α is a poor inducer of IL-12p70 production by DCs but increases IL-10 release [23, 26, 37, 39]. In addition, Ito et al. have shown that IFN-α-treated DCs induced regulatory T cells [37] and the effects were independent of the time of addition of IFN-α to the DC culture. The results obtained with IFN-β have been more consistent. IFN-β reduced IL-12 and increased IL-10 production by DCs derived from either MS patients or healthy controls [24,40–42].

The aim of the present study was to examine the ability of IFN-α2a (Roferon-A) to regulate the production of cytokines by DCs and T cells. IFN-α2a was chosen as it is used widely in clinical practice and here we show that, per se, it is sufficient to enhance DC maturation and to augment DC-mediated T cell proliferation. IFN-α2a increased production of IL-10 and IL-12 from DCs in response to CD40 ligation and lipopolysaccharide (LPS) stimulation, although LPS induced very little IL-12 release. As a consequence, the production of both IFN-γ and IL-5 by naive T cells in response to IFN-α2a-treated DC was also enhanced. In addition, the production of IL-12 was increased markedly by IFN-α2a-treated DCs when IL-10 was neutralized. In contrast, when IFN-α2a and LPS were acting simultaneously on DCs, IL-10 and IL-12 production, stimulated by CD40 ligation 36 h later, was inhibited, as well as IFN-γ and IL-5 release by the DC-stimulated T cells. These data suggest that IFN-α2a is sufficient to promote DC immunogenicity and to amplify a Th0 type of response. However, the presence of other stimuli can inhibit the effect of IFN-α2a. Altogether, these data may explain some of the divergent results seen in other studies and have important implications for the design of DC-based vaccines, where a Th1 type of response is sought.

Materials and methods

Media and reagents

DCs were cultured in X-VIVO 20 serum free medium (BioWhittaker, Walkersville, MD, USA) supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin, 2 mMl-glutamine and 2% human serum (HS). Mixed lymphocyte reactions (MLRs) were performed in RPMI-1640 (Life Technologies, UK) supplemented with 10% HS. The following human cytokines were used: granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4 (both from First Link, UK), LPS (from Escherichia coli; Sigma, UK), rhIFN-α2a (Roche Laboratories, Nutley, NJ, USA), rhIFN-γ (AL-Immuno Tools, Germany), prostaglandin E2 (PGE2) (Sigma, St Louis, MO, USA). Purification of peripheral blood CD4+ T cells was performed by using the following antibodies: anti-CD8 [American Type Culture collection (ATCC), USA], anti-CD14 (Sigma), anti-CD19 and anti-CD33 (Becton Dickinson, San Jose, CA, USA), anti-CD16, anti-CD56 and anti-CD45RO (Caltag, CA, USA). Flow cytometric analysis was performed using the following fluorescein isothyocyanate (FITC)-conjugated monoclonal antibodies (mAbs): IgG1 and IgG2a isotype controls (Pharmingen, San Diego, CA, USA), anti-HLA-DR (Sigma), anti-CD11c (Dako, Glostrop, Denmark), anti-CD40 (Pharmingen), anti-CD83 and anti-CD86 (both from Caltag). Enzyme-linked immunosorbent assay (ELISA) development kits were used to measure production of IL-10, IL-12 (both kits from Pharmingen), IFN-γ (MS Biotechnology, Abington, UK) and IL-5 (Biosource International, Camarillo, CA, USA).

Cell lines

Mouse L cells, both non-transfected and transfected with CD40L-transfectants (CD40L-Tx) (kind gift of Professor Gordon, University of Birmingham, UK), were grown and maintained in RPMI-1640 supplemented with 10% fetal calf serum (FCS) (SeraQ, Sussex, UK).

DC generation and culture

Peripheral blood mononuclear cells (PBMCs) were generated from whole blood or from buffy coats of healthy individuals by Ficoll-Paque density gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden). On day 0, monocytes were obtained from PBMCs as a cell fraction, following 2-h adherence to plastic of tissue culture flasks (Greiner, Essen, Germany) or to six-well culture plates (Costar Corning, Corning, NY, USA) at 37°C. GM-CSF and IL-4 were added to the cells every other day. DCs were matured and/or treated with cytokines on day 6, as described in the various experiments. After 36 h, the cells were counted and analysed for expression of various cell surface markers and/or irradiated (60 Gy) for use as stimulators in MLR. Cytokines were harvested at different time-points to detect production of IL-10 and IL-12.

In some experiments DCs were not co-cultured with T cells, but with CD40L-Tx. In these experiments, DCs (2 × 104 cells/well in six-well plates) were added to CD40L-Tx cells (5 × 104 cells/well) on day 6 of DC culture or 36 h after DCs were treated with different cytokines and/or maturation factors, as described in the different experiments.

Purification of CD4+ T cells from PBMCs

The non-adherent population of PBMCs was collected and roll-mixed with a mixture of purified mAbs for 30 min at 4°C and subsequently washed twice to remove excess antibodies. In order to deplete the antibody-bound cells, these cells were roll-mixed with magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and coated with goat anti-mouse Ig for 30 min at 4°C. Beads/mAbs-coated cells were then removed by passage through a magnetic column (miniMAC system, Miltenyi Biotech).

Immunofluorescence staining and flow cytometric analysis

A total of 105 DCs per staining sample were incubated separately with the indicated mAbs at 4°C for 30 min. For negative control staining, DCs were stained with isotype-matched control antibodies. Following staining, cells were washed twice with ice-cold phosphate buffered saline (PBS) supplemented with 2% FCS. Flow cytometric analysis was performed using FACSCalibur flow cytometer and CellQuest Software (Becton Dickinson, Oxford, UK).

Blocking assays

As indicated in some experiments, purified mouse anti-human IL-10 and IL-12 antibodies were used at concentrations of 10 and 5 µg/ml, respectively, in order to block production of the corresponding cytokines. These antibodies were added to day 6 DCs.

MLR

MLRs were carried out in 96-well round-bottom plates. DCs in a range of 102−105 cells/well (in triplicates) were co-cultured with naive CD4+ T cells (6 × 104 cells/well). Controls included DCs alone and T cells alone. On day 5, wells were pulsed with 1 µCi/well [3H]thymidine (Amersham Imternational, Amersham, UK). Thymidine incorporation was measured after 20 h by a liquid scintillation counter (Wallac, Turku, Finland).

Cytokine ELISA

PVC microtitre plates (Nalge Nunc International, Denmark) were coated overnight at 4°C with the primary antibody diluted in NaHCO2 (0·1 M, pH = 9·6). Next, the plates were washed three times in PBS/0·1%Tween (Sigma), and then blocked with PBS/1% bovine serum albumin (BSA) (Fluka BioChemika, Buchs, Switzerland) for 2 h at room temperature. After washing (×3), samples and standards (prepared in decreasing quadrupling dilution) were plated. Biotinylated mAb (diluted in PBS/0·1%Tween/1% BSA) was then added to all wells. After 2 h incubation at room temperature, the plates were washed as described above, and incubated with streptavidin peroxidase conjugate (Biosource International) in PBS/1% BSA/0·1% Tween for 45 min. Plates were then washed eight times and incubated with 1,3,5 trimethoxy benzene (TMB) single solution (Zymed, San Francisco, CA, USA) until a blue colour developed. Absorbance was measured at a wavelength of 450 nm on an ELISA reader.

Results

IFN-α2a induced maturation of DCs in the absence of maturation factors

The effects of different concentrations of IFN-α2a on the expression of CD11c, HLA-DR, CD86 and CD83 on monocyte-derived DCs were investigated. Some up-regulation of cell surface molecules was already observed with 102 U/ml of IFN-α2a (Fig. 1). However, the optimal phenotypic maturation was observed with 103 U/ml of IFN-α2a. In contrast, higher concentration (104 U/ml) induced down-regulation of CD11c and HLA-DR compared to untreated DCs. Treatment of DCs with IFN-γ and PGE2 that were used as controls [43,44] induced up-regulation of all the markers analysed (data not shown).

Fig. 1.

Fig. 1

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Interferon (IFN)-α2a induced maturation of dendritic cells (DCs). DCs were cultured with X-VIVO 20 supplemented with 2% human serum (HS), and granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 were added every other day. On day 6, DCs were treated with IFN-α2a at different concentrations. After 36 h, DCs were harvested and stained with monoclonal antibodies (mAbs) specific for different cell surface molecules (as indicated). Expression of these molecules by DCs was analysed by using flow cytometer. Filled curves − staining with the indicated antibodies, empty curves –staining with control antibodies. Upper numbers represent percentages of positive cells and lower numbers represent mean fluorescence intensity. The results are representative of five different experiments.

In parallel cultures, DCs were treated with different concentrations of IFN-α2a in the presence of LPS. The concentration of LPS selected was suboptimal and only marginal production of IFN-α by DCs was detected (data not shown). As shown in Fig. 2, LPS induced up-regulation of all the markers analysed and no marked phenotypic changes occurred with the addition of IFN-α2a. As seen with IFN-α2a alone, some inhibition of expression of CD11c and HLA-DR was observed with 104 U/ml of IFN-α2a compared to LPS alone. Similar results were obtained with IFN-γ and PGE2, so when they were added to DCs together with LPS no significant phenotypic changes were observed (data not shown).

Fig. 2.

Fig. 2

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Interferon (IFN)-α2a did not affect lipopolysaccharide (LPS)-mediated phenotypic maturation of dendritic cells (DCs). On day 6 of culture, DCs were treated with LPS (20 ng/ml) in the presence or in the absence of IFN-α2a at different concentrations. After 36 h, DCs were harvested and stained with monoclonal antibodies (mAbs) specific for different cell surface molecules (as indicated). Expression of these molecules by DCs was analysed by using flow cytometer. Filled curves − staining with the indicated antibodies, empty curves − staining with control antibodies. Upper numbers represent percentages of positive cells and lower numbers represent mean fluorescence intensity. The results are representative of four different experiments.

IFN-α2a enhanced the T cell stimulatory capacity of DCs

The stimulatory capacity of DCs treated with IFN-α2a was analysed in MLR. Purified CD4+CD45RA+ naive T cells were cultured with different numbers of allogeneic DCs and T cell proliferation was measured. Treatment of DCs with IFN-α2a enhanced T cell proliferation in a dose-dependent manner with the highest response to DCs treated with 103 U/ml of IFN-α2a (Fig. 3a).

Fig. 3.

Fig. 3

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Interferon (IFN)-α2a treatment of dendritic cells (DCs) enhanced T cell proliferation. On day 6 of culture, DCs were treated with different concentrations of IFN-α2a alone (a) or in the presence of lipopolysaccharide (LPS) (b). DCs were also treated with IFN-γ or with prostaglandin E2 (PGE2) alone (c) or together with LPS (d). After 36 h, a range of 102−105 DCs was co-cultured with allogeneic CD4+CD45RA+ T cells (6 × 104/cells/well) for 6 days. T cell proliferation was measured by [3H] incorporation and expressed as counts per minute (cpm). The results are representative of three independent experiments. IM = immature.

When DCs were treated with IFN-α2a together with LPS, no additional stimulatory impact on T cell proliferation was observed (Fig. 3b). While PGE2 treatment of DCs enhanced T cell proliferation, IFN-γ-treated DCs induced the same levels of proliferation as immature DCs. High numbers of DCs with both treatments inhibited T cell proliferation (Fig. 3c). When DCs were treated simultaneously with LPS and PGE2 or with IFN-γ, a marginal increase in T cell response was observed (Fig. 3d).

IFN-α2a amplified release of IL-10 and IL-12 from DCs

DCs in peripheral tissues release cytokines in response to ‘danger signals’. In order to see the influence of IFN-α2a on cytokine release by DCs, day 6 monocyte-derived DCs were cultured with IFN-α2a alone or in combination with different maturation/activation stimuli. Supernatants were harvested at different time-points and IL-12p70 and IL-10-productions were measured. The amount of cytokines produced by unstimulated DCs was minimal (less than 40 pg/ml), although some differences in IL-10 production were observed among the different culture conditions (data not shown). In contrast, ligation of CD40 on DCs induced release of IL-10 that was further amplified by the presence of IFN-α2a (Fig. 4b). IL-12 production was measured following CD40 ligation and the addition of IFN-α2a led to an increase in IL-12 production (Fig. 4a). When LPS was added to DCs, IL-10 production was detected and was increased further in the presence of IFN-α2a (Fig. 4d). However, IL-12 was induced only marginally by LPS stimulation of DCs (80 pg/ml) and IFN-α2a did not significantly augment IL-12 induction (100 pg/ml) (Fig. 4c). In contrast, the addition of IFN-γ markedly increased IL-12 production by DCs (Fig. 4c) and PGE2 increased IL-10 release (Fig. 4d).

Fig. 4.

Fig. 4

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Interferon (IFN)-α2a treatment of stimulated dendritic cells (DCs) amplified interleukin (IL)-10 and IL-12 production. On day 6 of culture, DCs were treated with IFN-α2a, IFN-γ and prostaglandin E2 (PGE2) in the presence of either CD40L-Tx (a, b) or lipopolysaccharide (LPS) (c, d). Supernatants were harvested at 16, 24 and 36 h and production of IL-12 (a, c) and IL-10 (b, d) was measured by enzyme-linked immunosorbent assay (ELISA). The results are representative of six different experiments.

IL-10 has been shown to regulate IL-12 production by DCs [45–47]. The possibility that IL-10 has an inhibitory effect on IL-12 production was examined by using neutralizing antibodies specific for IL-10. The results are shown in Fig. 5. The effect of IFN-α2a addition is comparable to Fig. 4. When a neutralizing antibody specific for IL-10 was added to DCs stimulated with either LPS or CD40L-Tx, IL-12 production was enhanced two- to threefold in both stimulatory conditions (Figs 5a,b). The IL-12 enhancing effect in the presence of anti-IL-10 was particularly evident when IFN-α2a was added to DCs stimulated with LPS compared to CD40L-Tx.

Fig. 5.

Fig. 5

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Interleukin (IL)-12 production by interferon (IFN)-α2a-treated dendritic cells (DC) was enhanced further in the presence of anti-IL-10 antibody. On day 6, DCs were stimulated with CD40L-Tx or with lipopolysaccharide (LPS) in the absence or in the presence of IFN-α2a. At the same time, anti-IL-10 or anti-IL-12 monoclonal antibodies (mAbs) were added to the different cultures and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA). The results are representative of three different experiments.

As opposed to the inhibitory effect of IL-10 on IL-12 production, IL-12 has not been shown to have a regulatory effect on IL-10 production [45]. As shown in Fig. 5b, the addition of anti-IL-12 neutralizing antibody with LPS resulted in no marked changes in the levels of IL-10 production by DCs. In contrast, some decrease in IL-10 production was observed when DCs were stimulated with CD40L-Tx at 24 h only.

IFN-γ and IL-5 production by T cells were amplified by treatment of the stimulating DCs with IFN-α2a

One of the key functions of DCs is priming of the immune response and directing the Th1/Th2 equilibrium. To prime T cells, DCs need to migrate from the peripheral tissues to the secondary lymphoid organs and it is there that DCs interact with naive T cells and dictate T helper polarization. To mimic this function of DCs in vitro, DCs that were pretreated with IFN-α2a for 36 h were co-cultured with naive allogeneic T cells.

DCs pretreated with IFN-γ and PGE2 were used as controls for Th1 and Th2 development, respectively. IFN-γ pretreatment of DCs induced IFN-γ production by T cells and PGE2 and IL-5 secretion [37, 43, 44, 48]. IFN-α2a-treated DCs demonstrated enhanced production of both IFN-γ (Fig. 6a) and IL-5 (Fig. 6b) by naive T cells. In contrast, when naive T cells were stimulated by DCs that were pretreated with IFN-α2a together with LPS, the production of IFN-γ (Fig. 6c) and IL-5 (Fig. 6d) by T cells was decreased.

Fig. 6.

Fig. 6

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Interferon (IFN)-γ and interleukin (IL)-5 production by T cells was amplified by IFN-α2a treatment of dendritic cells (DCs) in the absence of any previous stimulation. On day 6, DCs were treated with IFN-γ, IFN-α2a and prostaglandin E2 (PGE2) in the absence (a, b) or presence of LPS (c, d). After 36 h, different numbers of DCs were co-cultured with allogeneic CD4+CD45RA+ T cells for 6 days. On day 5, supernatant was harvested and production of IFN-γ and IL-5 was measured by enzyme-linked immunosorbent assay (ELISA). The results are representative of more than five different experiments.

A possible correlation between IFN-γ and IL-5 production by T cells with cytokine production by DCs was examined when CD40 was ligated on the same DCs preparations used to stimulate T cells and IL-10/IL-12 release was analysed. Both cytokines were enhanced by treatment of DCs with IFN-α in isolation (Fig. 7a,b). In contrast, when DCs were stimulated with IFN-α and LPS 36 h previously, the cytokine production was either unchanged or decreased compared with LPS treatment alone (Fig. 7c,d). As published previously, the treatment of DCs with PGE2 in the presence or absence of LPS, while amplified IL-10 production, inhibited IL-12-release. An opposite IL-10/IL-12 production pattern was obtained with DCs treated with IFN-γ [43,48].

Fig. 7.

Fig. 7

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Interferon (IFN)-α2a-treated dendritic cells (DCs) produced interleukin (IL)-10 and IL-12 after CD40 ligation in the absence of any previous stimulation. Day 6 DCs were treated with IFN-α2a, IFN-γ and prostaglandin E2 (PGE2) in the absence (a, b) or presence (c, d) of lipopolysaccharide (LPS). After 36 h, DCs were collected and co-cultured with CD40L-Tx. Production of IL-10 and IL-12 in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA) after 16, 24 and 36 h. The results are representative of four different experiments.

Discussion

The effects of type I IFNs on DC maturation and functions have been investigated extensively in vitro, often with conflicting results. This may be due to the variability of the protocols for generation and culture of DCs, the different timing of type I IFNs addition, as well as the use of different subtypes of type I IFN (reviewed in [49]). This study investigated the effect of one of the IFNs subtypes (IFN-α2a) that is used widely in the clinic.

IFN-α2a induced maturation of DCs and increased their allostimulatory capacity. IL-10 and IL-12 production by DCs upon ligation of CD40 was amplified by IFN-α2a treatment. In contrast, conditioning of DCs with IFN-α2a in the presence of other maturation factors, such as LPS, induced down-regulation of cytokine production by DCs and T cells. We have also found that when IL-10 is limiting, IFN-α2a enhanced IL-12 production by DCs. Thus, the environment in which IFN-α is present plays a central role in determining its effects on DC function. These observations may help to explain some of the conflicting results seen in previous studies regarding the effects of type I IFNs on DC maturation and function.

Type I IFNs have been shown to be key cytokines for DC development and functions. Mice lacking a functional receptor for type I IFNs or in vitro addition of neutralizing anti-IFN-αβ receptor abrogated DC maturation, IFN-γ production by DCs and their T cell stimulatory ability [50]. Furthermore, type I IFNs induced expression of both IL-15 and IL-15 receptor α-chain; and, IL-15 mediated activation of DCs was abolished in mice deficient for the IFN-αβ receptor [51].

The present study confirmed that IFN-α enhanced phenotypic maturation of DCs in a dose-dependent manner and increased their capacity to stimulate T cells [27,28]. These results are supported by a recent study by Pollara et al. [52] in which direct infection of monocyte-derived DCs by herpes simplex virus was responsible for DC activation. However, production of IFN-α from infected DCs induced DC maturation of bystander, non-infected DCs.

The treatment of DCs with IFN-α2a per se was not sufficient to induce cytokine production by DCs. Indeed, Ebner and colleagues have shown that appropriate maturation stimuli are required for IL-12 production by DCs, such as CD40 ligation or a bacterial signal [53]. In this study, IFN-α2a enhanced the production of both IL-12 and IL-10 by CD40-stimulated DCs. In the periphery, DCs encounter CD40L expressed on platelets that have been shown to express this ligand and hence exposure to CD40L in the periphery may occur in vivo [54,55]. These results are in line with other data using IFN-α2a, where significant production of cytokines by IFN-α-treated DCs required exposure to additional signals [27,28]. Recently, Mailliard et al. have shown that when IFN-α was added to a DC maturation cocktail, these DCs could produce vast amounts of IL-12 following subsequent ligation with CD40L-Tx [56]. Similarly to our study, this study demonstrated that DC maturation does not necessarily have to be associated with the exhaustion of their ability to produce IL-12, as suggested in previous studies [57,58]. In contrast, stimulation of DCs with LPS induced mainly production of IL-10 that was further amplified by IFN-α, as reported previously [25,59]. However, by neutralizing IL-10, a marked production of IL-12 was detected by LPS-stimulated DCs, as will be discussed later. The differential effect of IFN-α on DCs stimulated by either CD40 ligation or in the presence of LPS was confirmed recently using recombinant IFN-β [60].

The cytokines produced by DCs play an important role not only in peripheral tissues but also when DCs migrate to the T cell areas of lymph nodes, where they prime naive T cells. In our protocol, we used DCs 36 h after treatment with IFN-α, as it has been reported in the murine system that T cells form small clusters around DCs 24 h after the injection of an antigen [61,62]. We have shown that the enhancing effect of IFN-α2a on cytokine production by DCs was abolished when DCs were activated by both IFN-α and LPS before CD40 ligation. These results support a study by Heystek et al., indicating that IFN-α/β enhanced IL-12 production by immature DCs but inhibited IL-12p70 production by mature DCs [38]. Finally, when DCs were pretreated with LPS and then exposed to CD40L-Tx after 36 h, no detectable decrease in IL-12 was observed compared to CD40 ligation alone. These results seem to contradict previous work, however, in our system, very little or no IL-12 was detected in response to LPS [57,58]. The inhibition of T cell proliferation observed may be due to the production of inhibitory cytokines, such as TGF-β, or the up-regulation of molecules of the B7 family known to be involved in the down-modulation of the immune response [63]. Unlike IFN-α, pretreatment of DCs with IFN-γ and PGE2 further amplified IL-12 and IL-10 production by DCs, respectively, upon subsequent encounter with CD40L-Tx.

IL-10 is an anti-inflammatory cytokine that was demonstrated to inhibit proinflammatory cytokines such as IL-12 [47,64]. This inhibition has been associated with suppression of a Th1 response [65] and with the induction of regulatory T cells [66]. In the present study, IL-10 was found to regulate IL-12 production but not vice versa. This confirms a previous study showing that pretreatment of purified human monocytes with IL-10 suppressed IFN-α as well as IFN-γ production from these cells [67]. Furthermore, a recent study in human macrophages has shown that IFN-α can interfere with the activity of IL-10 by diminishing the suppressive effect of IL-10 on TNF-α production from LPS-stimulated macrophages [68].

In summary, we find that IFN-α2a is a potent immunostimulatory cytokine that enhances DC maturation and a Th0 type of response by acting on immature DCs and augmenting cytokine release. However, we find that other factors, such as bacterial products, can substantially modify the effects of IFN-α2a. It seems likely that the ultimate fate of the developing DCs when IFN-α2a is involved is determined by the presence or absence of other influencing factors. Thus, type I IFNs should perhaps be regarded as an immunological accelerator that modifies the magnitude of the immune response but does not alter its direction.

Acknowledgments

This work was financed in part by the Wellcome Trust Foundation.

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