Differential regulation of Th1 responses and CD154 expression in human CD4⁺ T cells by IFN-α

Abstract

Like interleukin (IL)-12, interferon (IFN)-α has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-α on CD154 expression in human CD4⁺ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4⁺ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-α and IL-12 in the complete absence of accessory cells. IFN-α suppressed CD154 protein and mRNA expression in CD4⁺ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-α by itself did not enhance IFN-γ production or mRNA expression in CD4⁺ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-α enhanced IL-12Rβ2 mRNA expression in anti-CD3-stimulated CD4⁺ T cells. Neither IFN-α nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4⁺ T cells. These results indicate that IFN-α by itself enhances CD154 expression in CD4⁺ T cells independently of the induction of IFN-γ mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-α might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4⁺ T cells might be regulated through different mechanisms.

INTRODUCTION

Interleukin (IL)-12 is a heterodimeric cytokine composed of 40-kDa (p40) and 35-kDa (p35) subunits, produced by macrophages, B cells, and dendritic cells (DCs) [1,2]. One of the most important roles of IL-12 in immune regulation is to induce the differentiation of Th1 cells from naive CD4⁺ T cells [3,4]. Previous studies showed that IL-12 up-regulates CD154 expression on human T cells at the protein level [5,6] as well as at the mRNA level [5]. It was therefore suggested that human Th1 responses driven by IL-12 might be associated with enhanced expression of CD154 [6]. However, it remains unclear whether the enhanced CD154 expression might be the sequelae of Th1 responses or a chanced association, as the precise mechanism of the enhancement of CD154 expression on human T cells by IL-12 has not been elucidated.

Interferon (IFN)-α is a member of a multigene family, which presents potent antiviral actions as well as immunoregulatory activities, such as the enhancement of cytotoxic activity of T cells and natural killer (NK) cells [7]. IFN-α is produced by macrophages, DCs and fibroblasts and its production is enhanced prominently during viral and bacterial infection [7]. The immune modulating functions of IFN-α suggest that IFN-α may be an important link between innate and adaptive immune responses [8]. Like IL-12, IFN-α has also been shown to activate Stat4 [9,10] and induce Th1 development directly in humans [10]. Because IL-12 induces Th1 responses as well as the enhancement of CD154 expression, it is possible that IFN-α might also influence CD154 expression in human T cells. In fact, several studies have demonstrated that IFN-α results in the development of lupus-like autoimmunity [11–13], in which abnormal expression of CD154 might be involved [14,15]. Thus, it is highly likely that IFN-α might enhance CD154 expression. However, the effects of IFN-α on CD154 expression in human T cells have not been elucidated.

The current studies were therefore undertaken to explore the effects of IFN-α on CD154 expression in activated human CD4⁺ T cells, utilizing a system with immobilized anti-CD3, which permits stimulation of all peripheral blood T cells in the complete absence of accessory cells. This system therefore enables us to evaluate the effects of IFN-α on CD4⁺ T cells without any effects of endogenous IL-12 produced by accessory cells or B cells. Special attention was directed to the relationship between CD154 expression and induction of Th1 responses.

MATERIALS AND METHODS

Monoclonal antibodies (MoAbs) and reagents

A variety of MoAbs were used, including 64·1 (a gift of Dr P. E. Lipsky, National Institute of Health, Bethesda, MD, USA), an IgG2a MoAb directed at the CD3 molecule on mature T cells; fluorescein isothiocyanate (FITC)-conjugated anti-CD154 (a murine IgG1 MoAb, clone 24–31) (Ancell, Bayport, MN, USA); phycoerythrin (PE)-conjugated anti-IL-2 receptor α-chain (CD25) (a murine IgG2a MoAb, clone 1HT44H3) (Immunotech, Marseille, France); FITC- and PE-conjugated control mouse IgG1 and IgG2a (Dako, Glostrup, Denmark); goat polyclonal IgG anti-human IL-12 (R&D Systems, Minneapolis, MN, USA); anti-CD28 MoAb (a murine IgG1 MoAb, clone CD28·2) (Immunotech); a murine IgG1 control MoAb, MOPC 21 (Cappel, West Chester, PA, USA); and goat control IgG (Cappel). Recombinant human IL-12 was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Recombinant human IFN-α 2a was a gift of Nippon Roche (Tokyo, Japan). A metalloproteinase inhibitor KB8301 was purchased from PharMingen (San Diego, CA, USA). Actinomycin D was purchased from Sigma Chemical Co. (St Louis, MO, USA).

Culture medium

RPMI-1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with penicillin G 100 U/ml, streptomycin 100 µg/ml, L-glutamine 0·3 mg/ml, and 10% FBS (Life Technologies) was used for all cultures.

Cell preparation

Peripheral blood mononuclear cells (PBMC) were obtained from healthy adult volunteers by centrifugation of heparinized venous blood over sodium diatrizoate-Ficoll gradients (Histopaque; Sigma). PBMC were depleted of monocytes and NK cells by incubation with 5 mM L-leucine methyl ester HCl (Sigma) in serum-free RPMI-1640, as described elsewhere [16]. T cells were obtained from the treated cell population by rosetting with neuraminidase-treated sheep red blood cells (SRBC) as described previously [6]. Purified CD4⁺ T cells were prepared further by positive selection with anti-CD4 microbeads and MACS (Miltenyi Biotec, Auburn, CA, USA). The CD4⁺ T cell population obtained in this manner contained <0·1% esterase-positive cells, <0·1% NK cells, <0·1% CD19⁺ cells, and>96% CD4⁺ T cells.

Cell culture techniques for induction of the expression of CD154

Anti-CD3 MoAb, 64·1 was diluted in RPMI-1640 (2 µg/ml), and 50 µl were placed in each well of 96-well flat-bottomed microtitre plates (no. 3596; Costar, Cambridge, MA, USA) and incubated at room temperature for 1 h [17]. Purified CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 with or without IFN-α and IL-12. KB8301 was dissolved in dimethylsulphoxide (DMSO), and added to cultures at a final concentration of 10 µM with 0·01% DMSO to prevent the cleavage of CD154 from the membranes [6]. The cells were incubated for 1–5 days at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Immunofluorescence staining of cell surface markers and analysis by flow cytometry

After the incubation, the cells were washed once with phosphate-buffered saline (PBS) containing 2% normal human serum and 0·1% sodium azide (staining buffer). The cells were reacted in suspension by incubating for 30 min at 4°C with saturating concentrations of FITC- or PE-conjugated MoAbs. After the cells were washed twice with staining buffer, they were fixed with 1% paraformaldehyde in PBS pH 7·4 for more than 5 min at room temperature. The cells were analysed using an EPICS XL flow cytometer (Coulter) equipped with an argon-ion laser at 488 nm, as described previously [6]. The percentages of cells staining positively for each MoAb were determined by integration of cells above a specified fluorescence channel, calculated in relation to isotype-matched control MoAb. Density of staining was expressed as the change in mean fluorescence intensity (MFI) for staining with the MoAb of interest calculated by subtracting the MFI of staining with the isotype-matched control MoAb.

RNA isolation and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from cultured cells using the Trizol reagent (Life Technologies) according to the manufacturer's application protocol and quantified spectrophotometrically. cDNA samples were prepared from 1 µg of total RNA using the SuperScript reverse transcriptase preamplification system (Life Technologies) with oligo (dT) primer and subjected to PCR reactions using the following primers: sense, 5′-GGCCATTATGCA CAGGTTGAAT-3′ and antisense, 5′-GGGGAGGGAAGAG ACTGACAAA-3′for CD154 (387 base pairs (bp)); sense, 5′-AGTTATATCTTGGCTTTTCA-3′ and antisense, 5′-ACCG AATAATTAGTCAGCTT for IFN-γ (356 bp); sense, 5′-GAGG GACTGGTACTGCTTAATCG-3′ and antisense, 5′-CCCTGCC TCACACAGGTTCA-3′for IL-12Rβ2 chain (516 bp); and sense, 5′-ATGGCCACGGCTGCTTCCAGC-3′and antisense, 5′-CAGGAGGAGCAATGATCTTGAT-3′ for β-actin (321 bp) as a control. Samples were amplified by AmpliTaq Gold (Perkin Elmer, Emeryville, CA, USA). PCR reactions were carried out as follows: denaturation at 94°C for 30 s; annealing at 58°C (CD154) or 55°C (IFN-γ, IL-12Rβ2 and β-actin) for 30 s; and extension at 72°C for 1 min. After optimal cycles for each PCR reaction, extension was continued at 72°C for additional 10 min. PCR products were analysed by electrophoresis on 1·5% agarose gels and visualized by ethidium bromide staining. Band intensities were then quantified and analysed with NIH image version 1·62. All results were calibrated to the β-actin band intensity amplified from the same cDNA sample. The identity of each PCR product was verified by sequence analysis.

mRNA stability studies

Purified CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 with or without IFN-α and IL-12. After 48 h or 96 h of incubation, actinomycin D (10 µg/ml) was added to the cultures. After various times from the addition of actinomycin D, the cells were harvested and total RNA was isolated. cDNA samples were then prepared and subjected to PCR reactions for detection of CD154 and β-actin as described above.

Measurement of IFN-γ and IL-12

IFN-γ contents in the culture supernatants were measured using a solid phase enzyme-linked immunosorbent assay (ELISA) as described previously [6]. IL-12 contents in the supernatants were measured using an ELISA kit, Cytoscreen (BioSource International, Camarillo, CA). The detection limit of the assay was approximately 0·8 pg/ml of IL-12.

Statistical analysis

The results were analysed for statistical significance by paired sample t-test.

RESULTS

IFN-α exerts biphasic effects on CD154 expression of immobilized anti-CD3-activated CD4⁺ T cells depending on the state of activation

Figure 1 shows the time kinetics of CD154 protein expression on immobilized anti-CD3-activated CD4⁺ T cells with or without IFN-α and IL-12. CD154 expression was not detected on unstimulated CD4⁺ T cells (data not shown), but was induced after 24 h of stimulation with immobilized anti-CD3. IFN-α significantly suppressed CD154 expression at 24 h of culture irrespective of the presence of IL-12 (Fig. 1b). The effect of IL-12 on the enhancement of CD154 expression was not significant at 24 h of stimulation, as was consistent with previous studies [6]. The expression of CD154 as well as its enhancement by IL-12 was significant at 72 h of stimulation with immobilized anti-CD3, at which time IFN-α still suppressed the expression of CD154, although the suppressive effects appeared to be less marked (Fig. 1b). Between 72 h and 120 h of cultures, a remarkable decline in CD154 protein expression was observed, as was also reported previously [6]. Of note, however, IFN-α significantly enhanced the expression of CD154 on immobilized anti-CD3-stimulated CD4⁺ T cells at 120 h of cultures (Fig. 1b). The enhancement of CD154 expression was dose-dependent between 100 IU/ml and 1 × 10⁵ IU/ml of IFN-α (data not shown). IL-12 also enhanced the expression of CD154 on CD4⁺ T cells at 120 h of cultures. Of note, IFN-α further enhanced the expression of CD154 on activated CD4⁺ T cells even in the presence of saturating concentrations of IL-12 (10 ng/ml) (Fig. 1b). These results indicate that IFN-α exerts biphasic effects on the expression of CD154 on human CD4⁺ T cells depending on the state of activation irrespective of the presence of IL-12.

Fig. 1.

Time kinetics of CD154 protein expression on immobilized anti-CD3-activated CD4⁺ T cells: effects of IFN-α and IL-12. CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64·1, 100 ng/well) in the presence of a metalloproteinase inhibitor KB8301 (10 µM) with or without IFN-α (1 × 10⁵ IU/ml) and IL-12 (10 ng/ml). After various periods of incubation, the cells were harvested and stained with FITC-;conjugated anti-CD154 (open histogram) MoAb or control MoAb (closed histogram). (a) Percentage of positive and mean fluorescence intensity (MFI) of specific staining for CD154 are indicated. (b) MFI of six independent experiments are indicated. Each line on the graph is representative of the same cell preparation from the same donor. *P < 0·05.

The next experiments examined the effects of IFN-α on CD154 mRNA expression in immobilized anti-CD3-activated CD4⁺ T cells, using semiquantitative RT-PCR. As shown in Fig. 2a, CD154 mRNA expression was not detected in unstimulated CD4⁺ T cells. The enhancement of CD154 mRNA expression by IL-12 was observed as early as 24 h, and continued throughout the cultures up to 120 h (Fig. 2b). By contrast, IFN-α down-regulated CD154 mRNA expression significantly irrespective of the presence of IL-12 at 24 h of stimulation (Fig. 2b). However, IFN-α enhanced the expression of CD154 mRNA in anti-CD3-activated CD4⁺ T cells significantly at 120 h of cultures, even in the presence of IL-12 (Fig. 2b). These results indicate that IFN-α also exerts biphasic effects on CD154 mRNA expression, depending on the state of activation of immobilized anti-CD3-stimulated CD4⁺ T cells, whereas IL-12 constantly enhances CD154 mRNA expression in anti-CD3-stimulated CD4⁺ T cells throughout the cultures for as long as 120 h.

Fig. 2.

Time kinetics of CD154 mRNA expression in immobilized anti-CD3-activated CD4⁺ T cells: effects of IFN-α and IL-12. (a) CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64·1, 100 ng/well) with or without IFN-α (1 × 10⁵ IU/ml) and IL-12 (10 ng/ml). After 24 h or 120 h of incubation, total RNA was isolated and RT-PCR was performed using specific primers for CD154 and β-actin. Lane 1: unstimulated CD4⁺ T cells; lane 2, 6: nil; lane 3, 7: IFN-α; lane 4, 8: IL-12; lane 5, 9: IFN-α + IL-12; M: marker. (b) Densitometric measurement of CD154 mRNA expression. All results were calibrated to the β-actin band intensity. Each line on the graph is representative of the same cell preparation from the same donor. *P < 0·05.

IFN-α directly enhances CD154 expression in immobilized anti-CD3-activated CD4⁺ T cells independently of the induction of Th1 responses

It was shown that IL-12Rβ2 subunit, a binding and signal transducing component of IL-12R, is critical for functional IL-12R expression and induction of high affinity IL-12 bindings [18]. As shown in Fig. 3, IFN-α enhanced significantly the expression of IL-12Rβ2 mRNA in immobilized anti-CD3-;stimulated CD4⁺ T cells at 24 h of cultures, as is consistent with the previous report [19]. Moreover, the enhancement of IL-12Rβ2 mRNA expression by IFN-α continued throughout the cultures for up to 120 h (data not shown). The data therefore raise the possibility that IFN-α might enhance CD154 expression on anti-CD3-activated CD4⁺ T cells through mechanisms depending on IL-12. However, IL-12 could not be detected in the culture supernatants throughout the cultures of CD4⁺ T cells for as long as 120 h (data not shown). Moreover, neutralizing anti-IL-12 antibody did not influence the IFN-α-mediated enhancement of CD154 expression on CD4+ T cells stimulated with immobilized anti-CD3 for 120 h, whereas it abrogated completely the enhancement of the expression of CD154 by IL-12 (Table 1). These results therefore obviate the possibility that the enhancement of CD154 expression of anti-CD3-stimulated CD4⁺ T cells by IFN-α might be mediated by the action of IL-12 and thus demonstrate that IFN-α by itself enhances CD154 expression on CD4⁺ T cells independently of IL-12.

Fig. 3.

IL-12Rβ2 mRNA expression in immobilized anti-CD3-activated CD4⁺ T cells: effects of IFN-α and IL-12. (a) CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64·1, 100 ng/well) with or without IFN-α (1 × 10⁵ IU/ml) and IL-12 (10 ng/ml). After 24 h of incubation, total RNA was isolated and RT-PCR was performed using specific primers for IL-12Rβ2 and β-actin. Lane 1: unstimulated CD4⁺ T cells; lane 2: nil; lane 3: IFN-α; lane 4: IL-12; lane 5: IFN-α + IL-12; M: marker. (b) Densitometric measurement of IL-12Rβ2 mRNA expression. All results were calibrated to the β-actin band intensity. Each line on the graph is representative of the same cell preparation from the same donor. *P < 0·05.

Table 1.

Effect of a neutralizing anti-IL-12 on the enhancement of CD154 expression by IFN-α or IL-12

	CD154 expression
	Control IgG		Anti-IL-12
Cytokines	Positive (%)	MFI	Positive (%)	MFI
Nil	57·2	1·496	60·2	1·607
IFN-α	81·3	3·114	82·8	3·289
IL-12	73·5	2·357	58·2	1·558
IFN-α + IL-12	90·1	4·893	86·2	3·547

CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64·1, 100 ng/well) in the presence of a metalloproteinase inhibitor KB8301 (10 µM) with or without IFN-α (1 × 10⁵ IU/ml) and IL-12 (1 ng/ml). Goat polyclonal anti-IL-12 (10 µg/ml) or control goat IgG (10 µg/ml) was added as indicated. After 120 h of incubation, the cells were harvested and stained with FITC-conjugated anti-CD154 MoAb or control MoAb, followed by analysis on flow cytometry. Percentage of positive and mean fluorescence intensity (MFI) of specific staining for CD154 are indicated.

As shown in Fig. 4, IFN-α did not enhance IFN-γ production and mRNA expression of anti-CD3-stimulated CD4⁺ T cells, whereas IL-12 significantly enhanced it. Of note, IFN-α did not suppress IFN-γ protein and mRNA expression in CD4⁺ T cells stimulated with anti-CD3 for 24 h. Of more importance, at 120 h of cultures IFN-α significantly enhanced IFN-γ expression only in the presence of exogenous IL-12 (Fig. 4a,c), although IFN-α enhanced CD154 expression even in the absence of exogenous IL-12 (Figs 1b and 2b). These results indicate that the enhancement of IFN-γprotein and mRNA expression of anti-CD3-stimulated CD4⁺ T cells by IFN-α requires the presence of IL-12, whereas the enhancement of CD154 expression by IFN-α does not. In this regard, it is most likely that CD154 expression and IFN-γ production in human CD4⁺ T cells might be regulated differently by IFN-α.

Fig. 4.

Time kinetics of IFN-γ protein production and mRNA expression of immobilized anti-CD3-activated CD4⁺ T cells: effects of IFN-α and IL-12. CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64·1, 100 ng/well) with or without IFN-α (1 × 10⁵ IU/ml) and IL-12 (10 ng/ml). (a) After various periods of incubation, the supernatants were assayed for IFN-γ content by ELISA. Each line on the graph is representative of the same cell preparation from the same donor. *P < 0·05. (b) After 24 h or 120 h of incubation, total RNA was isolated and RT-PCR was performed using specific primers for IFN-γ and β-actin. Lane 1: unstimulated CD4⁺ T cells; lane 2, 6: nil; lane 3, 7: IFN-α; lane 4, 8: IL-12; lane 5, 9: IFN-α + IL-12; M: marker. (c) Densitometric measurement of IFN-γ mRNA expression. All results were calibrated to the β-actin band intensity. Each line on the graph is representative of the same cell preparation from the same donor. *P < 0·05.

It has been shown that optimal activation of T cells requires signalling through CD28 co-stimulation [20]. It was therefore possible that the lack of enhancement of IFN-γ expression by IFN-α might be due to the absence of CD28 co-stimulation signals. To address this point, experiments were undertaken in which CD4⁺ T cells were stimulated with immobilized anti-CD3 with or without IFN-α and IL-12 for 120 h in the presence of anti-CD28 MoAb or control MoAb. As can be seen in Fig. 5, CD28 co-stimulation enhanced significantly the expression of CD154 mRNA and IFN-γ mRNA, as is consistent with previous reports [20,21]. Of note, even in the presence of CD28 co-stimulation, IFN-α did not enhance IFN-γ mRNA expression, unless IL-12 was supplemented. By contrast, IFN-α further enhanced CD154 mRNA expression in the presence of CD28 co-stimulation irrespective of the presence of IL-12. These results confirm that enhancement of IFN-γ expression in anti-CD3-stimulated CD4⁺ T cells by IFN-αrequires the presence of IL-12, but not that of CD28 co-stimulation.

Fig. 5.

Effect of IFN-α and IL-12 on the expression of CD154 mRNA and IFN-γ mRNA in immobilized anti-CD3-stimulated CD4⁺ T cells: effect of CD28 co-stimulation. CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64·1, 100 ng/well) with or without IFN-α(1 × 10⁵IU/ml) and IL-12 (10 ng/ml). Anti-CD28 MoAb or control IgG1 MoAb (4 µg/ml) was added as indicated. After 120 h of incubation, total RNA was isolated and RT-PCR was performed using specific primers for CD154, IFN-γ and β-actin. All results were calibrated to the β-actin band intensity. #P < 0·05 compared with cultures with control MoAb in the presence or absence of the same cytokines. Each line on the graph is representative of the same cell preparation from the same donor. *P < 0·05.

IFN-α and IL-12 do not influence the stability of CD154 mRNA in immobilized anti-CD3-activated CD4⁺ T cells

Final experiments examined whether the enhancement of CD154 mRNA expression in anti-CD3-stimulated CD4⁺ T cells by IFN-α and IL-12 results from an increase in the stability of CD154 mRNA. As shown in Fig. 6, IFN-α and IL-12 had no significant effects on the decay rate of CD154 mRNA in CD4⁺ T cells stimulated with immobilized anti-CD3 for 48 h or for 96 h. The results indicate that IFN-α and IL-12 do not influence the stability of CD154 mRNA in anti-CD3-activated CD4⁺ T cells.

Fig. 6.

IFN-α and IL-12 do not influence the stability of CD154 mRNA of CD4⁺ T cells activated with immobilized anti-CD3. CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64·1, 100 ng/well) with or without IFN-α (1 × 10⁵ IU/ml) and IL-12 (10 ng/ml). After 48 h or 96 h of incubation, actinomycin D (10 µg/ml) was added to the cell cultures. After various times from the addition of actinomycin D, the cells were harvested and total RNA was isolated. Semiquantitative RT-PCR was performed using specific primers for CD154 and β-actin. All results were calibrated to the β-actin band intensity. The data are expressed as the mean ± s.d. (error bars) of three independent experiments. The Y-axis denotes the percent of the CD154/β-actin ratio at the addition of actinomycin D. ◊, Nil; •, IFN-α; ▵, IL-12; ♦, IFN-α + IL-12.

DISCUSSION

The current studies investigated in detail the effects of IFN-α on CD154 expression of immobilized anti-CD3-stimulated CD4⁺ T cells. Intriguingly, IFN-α exerted biphasic effects on the expression of CD154, depending on the state of activation of immobilized anti-CD3-stimulated CD4⁺ T cells. Thus, IFN-α suppressed CD154 protein and mRNA expression in the initial phase of activation of CD4⁺ T cells, whereas IFN-α enhanced it at subsequent maturation phase. Because IFN-α suppressed CD25 expression on immobilized anti-CD3-activated CD4⁺ T cells at 24 h of culture (data not shown), it was possible that IFN-α might result in global inhibition of mRNA transcription/translation at the initial activation of CD4⁺ T cells. However, IFN-α enhanced IL-12Rβ2 mRNA expression in anti-CD3-activated CD4⁺ T cells at 24 h of cultures, as is consistent with previous studies [19]. Moreover, IFN-α did not suppress IFN-γ protein and mRNA expression in the initial activation phase. These results suggest therefore that the suppression of CD154 expression by IFN-α at the initial activation might not a result from global inhibition of mRNA transcription/;translation, but be a more specific event, although the precise mechanisms remain to be elucidated.

IFN-α as well as IL-12 enhanced CD154 protein and mRNA expression at 120 h of cultures in CD4⁺ T cells. Of note, IFN-α further enhanced CD154 mRNA expression in activated CD4⁺ T cells even in the presence of saturating concentrations of IL-12, indicating that IFN-α enhances CD154 expression in CD4⁺ T cells through mechanisms different from those of IL-12. It may be argued that IFN-α might enhance CD154 expression through up-regulation of the reactivity to IL-12, as IFN-α enhanced the expression of IL-12Rβ2 mRNA in anti-CD3-stimulated CD4⁺ T cells. However, no IL-12 could be detected in the culture supernatants of anti-CD3-stimulated CD4⁺ T cells, reflecting that there was no contamination of monocytes and B cells in the cultures. Moreover, neutralizing anti-IL-12 antibodies did not affect the enhancement of CD154 protein expression by IFN-α, although they totally blocked the enhancing effects of IL-12. The results therefore confirm that IFN-α by itself up-regulates CD154 expression in CD4⁺ T cells independently of IL-12.

Several studies have reported that type 1 IFNs (IFN-α/β) act directly on human, but not mouse, T cells to drive Th1 development, bypassing the need for IL-12-induced signalling [10]. However, the present study demonstrated that IFN-α did not affect IFN-γ mRNA and protein expression in CD4⁺ T cells, unless IL-12 was present, suggesting that IFN-α by itself might not be sufficient for the optimal induction of Th1 responses. It should be pointed out that CD4⁺ T cells were activated in the complete absence of accessory cells in the current study, whereas IFN-α induced Th1 responses in the presence of accessory cells in the previous studies [10,19,22]. It has been found that accessory cells provide co-stimulation signals as well as IL-12. It was therefore possible that the inability of IFN-α to enhance IFN-γ expression in anti-CD3-stimulated CD4⁺ T cells might be due to the absence of co-stimulation signals. In the present study, however, IFN-α did not enhance IFN-γ mRNA in anti-CD3-stimulated CD4⁺ T cells even in the presence of CD28 co-stimulation, unless IL-12 was supplemented, confirming that the presence of IL-12 was essential for the up-regulation of IFN-γexpression by IFN-α. It was therefore most probable that IFN-αinduced Th1 responses through up-regulation of the responsiveness to IL-12 secreted from accessory cells in those studies [10,19,22]. Accordingly, a recent study also suggests that IFN-α might enhance IFN-γ production in human T cells by IL-12-dependent mechanisms [23].

Of note, like IL-12, IFN-α has been shown to activate Stat4 [9,10]. However, a recent study has also suggested that activation of Stat4 may not be a sufficient factor for Th1 responses although it is a necessary factor [24]. Moreover, IFN-α can activate Stat4 not only in human Th1, but also in Th2 cells, whereas restimulation of human Th2 lines in the presence of IFN-α does not induce the production of IFN-γ[10]. Collectively, these data suggest the involvement of an important factor other than Stat4 in the expression of IFN-γ mRNA in human T cells. Consistently, recent studies disclosed that mitogen-activated protein kinases (MAPKs) may be involved in a Stat4-independent pathway of IL-12 responsiveness in human T cells [25]. Further studies are required for a complete understanding of the mechanisms of IL-12 signalings that lead to maximal Th1 responses.

A recent study has identified a protein complex which binds to a highly CU-rich portion of the CD154 3′ untranslated region (3′ UTR) and enhances the stability of CD154 mRNA in anti-CD3-activated T cells [26]. Of note, this protein complex was induced in extracts from 24 h and 48 h anti-CD3-activated T cells [26]. As the enhancement of CD154 mRNA by IFN-α and IL-12 was marked later in cultures, it was possible that IFN-α or IL-12 might increase CD154 mRNA expression through up-regulation of such a protein complex. However, neither IFN-α nor IL-12 influenced the stability of CD154 mRNA in 48 h and 96 h anti-CD3-activated CD4⁺ T cells. It is therefore unlikely that these cytokines might increase the expression of such binding proteins. It is suggested, rather, that IFN-α and IL-12 might enhance CD154 mRNA expression in anti-CD3-activated CD4⁺ T cells by up-regulating its transcription, although further studies will be required to confirm this point.

IFN-α is produced by macrophages, DCs and fibroblasts and its production is prominently enhanced during viral and bacterial infection [7]. In this study, the enhancement of CD154 expression on activated CD4⁺ T cells was observed at a dose as little as 100 IU/ml and its expression increased in a dose dependent manner (data not shown). Of note, high serum levels of endogenous IFN-α, which surpasses 2 × 10⁴ IU/ml, are observed in many cases of serious viral infections in humans [27]. It is therefore suggested that IFN-α in vivo might contribute to a variety of immune responses through enhancement of CD154 expression in activated CD4⁺ T cells.