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Keywords:

  • dendritic cells;
  • multiple sclerosis;
  • IFN-β;
  • IL-10

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-γ and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-γ by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-β and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-β and IL-10 significantly suppressed IFN-γ production by MNCs. IFN-β in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-β and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dendritic cells (DCs) as the most potent antigen-presenting cells (APCs) are critical controllers of the immune system, especially of T cell responses [1,2]. DCs reside in peripheral tissues and express major histocompatibility complex (MHC) class II and costimulatory molecules like CD80 and CD86, and are highly efficient in taking up antigen [3]. DCs process foreign proteins or antigens into short peptides that are bound to MHC. MHC-peptide complexes serve as ligands for T cell receptors (TCR) and deliver proper instruction to T cells. DCs provide T cells not only with antigen-MHC complexes and costimulatory signals which are required for activation of T cells [2–4], but also with a polarizing signal which provokes either T helper type 1 (Th1) or Th2 responses [5,6].

Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), is assumed to result from autoaggressive T cell-mediated immune responses to myelin antigens, e.g. myelin basic protein (MBP) [7,8]. Circulating myelin-reactive T cells undergo clonal activation and expansion, and accumulate within the CNS in MS [9,10]. Th1 cells producing, e.g. IFN-γ are considered to play a role in the inflammation, which initiates demyelination, axonal loss and ultimately formation of gliotic scar tissue throughout the CNS in MS [11,12]. Whether the phenotype and function of DCs are altered in MS, thereby affecting Th1 response, is not known.

IFN-β as an immunomodulatory cytokine is effective for the treatment of MS. IFN-β has been proved to reduce relapse rate and slow disease progression [13] as well as reduce disease activity and disease burden as reflected by magnetic resonance imaging (MRI) of the CNS [14]. However, IFN-β is not a cure for MS and its modes of action in MS remain to be clarified. It has recently been hypothesized that IFN-β-mediated suppression of IL-12 production by APCs may play a role for the beneficial effects of IFN-β in MS [15]. Better understanding of the action of IFN-β could promote the search for more effective agents or combination therapies and increase our knowledge about MS pathogenesis.

The present study was undertaken to investigate DC phenotype and function with focus on DC-induced T cell responses in MS. In vitro effects of IFN-β on DC functions were examined with respect to IL-12 production, costimulatory property in mixed leucocyte reaction (MLR) and induction of Th responses.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients and healthy controls

Twenty patients (14 females) with clinically definite MS, MS-like lesions on MRI and oligoclonal IgG bands in cerebrospinal fluid (CSF) were studied. Their age was 44 ± 12 years. The duration of MS was 3–30 years (mean = 12). Disability scores, as assessed by the Expanded Disability Status Score (EDSS) [16], varied between 2·5 and 7. Eleven of the patients had relapsing-remitting MS and 9 were examined during the secondary chronic progressive phase of MS. Ten of the patients were examined during ongoing treatment with IFN-β1a or IFN-β1b. The remaining patients with MS had never been treated with any immunomodulatory drugs, including corticosteroids. Controls consisted of 20 healthy subjects (14 females). Their age was 42 ± 11 years.

Culture medium and reagents

RPMI 1640 (Gibco, Paisley, UK) was used as medium, supplemented with 2 mm l-glutamine, 1% nonessential amino acids, 10% FCS, 50 U/ml penicillin and 50 µg/ml streptomycin (all from Gibco). Recombinant human (rh) GM-CSF (Leucomax; Novartis, Basel, Switzerland) and IFN-β-1b (Betaferon; Schering AG, Berlin, Germany) prepared for therapeutical purposes were used. Rh IL-4 was purchased from Genzyme (Cambridge, MA). Rh IL-10 and rh TNF-α were from R & D System (Minneapolis, MN).

Cell preparations and cultures

MNCs were isolated from heparinized blood by centrifugation on a discontinuous density gradient (Lymphoprep, 1·077 g/ml; Nycomed, Oslo, Norway). Immature DCs were then generated from adherent MNCs [2,17]. MNCs were plated in 6-well plates (Costar, Cambridge, MA) in 2 ml of culture medium. After 1·5 h incubation at 37°C and 5% CO2, the nonadherent cell fraction was removed. Adherent cells were cultured in the medium containing rhGM-CSF (final concentration 800 U/ml) and rhIL-4 (500 U/ml). Every other day, 0·5 ml medium was removed and the same volume of fresh medium containing GM-CSF + IL-4 was added. After 7 days of culture, purity of the DCs was > 85% as determined by flow cytometry (Becton Dickinson, Mountain View, CA) examining expression of CD3 (T cells), CD19 (B cells), CD56 (NK cells), CD14 (monocytes) and DC surface molecules CD1a, CD80, CD86 and HLA-DR (Fig. 1).

image

Figure 1. Representative flow cytometry data on phenotype of blood dendritic cells (DCs) from a healthy subject. Blood adherent mononuclear cells (MNCs) were cultured in the presence of GM-CSF and IL-4 for 7 days and analysed by flow cytometry. DCs lack the lineage markers CD3 (T cells), CD19 (B cells), CD56 (NK cells) and CD14 (monocytes), but express CD80, CD86, CD1a and HLA-DR. The appropriate isotype controls are presented by dotted lines.

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On day 7, DCs were resupended and cultured in 24-well flat-bottom plates (Costar) in the absence or presence of IL-10 (50 ng/ml), IFN-β (10–10 000 U/ml), TNF-α (50 ng/ml) or IL-10 (50 ng/ml) + IFN-β (1000 U/ml) for an additional 48 h.

Flow cytometry analysis

DCs were examined by flow cytometry and analysed with CellQuest Software (Becton Dickinson). The following monoclonal antibodies (mAbs) were used for staining: PerCP-conjugated anti-CD3 (T cells) (SK7, IgG1) and PE-conjugated anti-HLA-DR (SK10, IgG1) from Becton Dickinson; FITC-conjugated anti-CD1a (HI149, IgG1), PE-conjugated anti-CD14 (monocytes) (M5E2, IgG2a), FITC-conjugated anti-CD19 (B cells) (IV B193, IgG1), PE-conjugated anti-CD56 (NK cells) (B159, IgG1), PE-conjugated anti-CD80 (BB1, IgG1) and FITC-conjugated anti-CD86 (FUN-1, IgG1) from PharMingen. As controls, cells were stained with corresponding isotype-matched control mAbs.

Mixed leucocyte reaction and cytokine determination

After 7-day culture with GM-CSF + IL-4, DCs were incubated with or without cytokines under study for another 48 h. DCs were irradiated (15 Gy from a 137Cs source) and used as stimulators. 1 × 104 irradiated DCs as stimulators were plated in 96-well round-bottom plates (Nunc, Roskilde, Denmark) in triplicate and overlaid with 2 × 105 allogeneic MNCs from healthy donors as responders. After 72 h of coculture, the cells were incubated with [3H] thymidine (Amersham Life Science, Buckinghamshire, UK) at a concentration of 1 µCi/well for 18 h, and then transferred onto glass fibre filtres (Skatron, Lier, Norway). [3H] thymidine incorporation was measured using a beta-scintillation counter (Beckman, Fullerton, CA).

For determination of cytokine production by DCs, supernatants of DCs were harvested after 7 days' culture and tested in triplicate for IL12 p40 production by enzyme-linked immunosorbent assays (ELISA) according to the manufacturer's protocols (kits from PharMingen, San Diego, CA). MNCs were cocultured with allogeneic DCs. After 48 h, supernatants were harvested and analysed for IL-2, IL-4 and IFN-γ production using ELISA (PharMingen). Optical densities were measured using an ELISA reader.

In vitro assays of functional consequences of anti-CD80 and anti-CD86 mAbs

To determine a role of the costimulatory molecules CD80 and CD86 on the capacity of DC priming Th cell responses, the following mAbs were added to the cultures of DCs on day 7: anti-CD80 (10 µg/ml, IgG1, Serotec, Oxford, UK) or anti-CD86 (10 µg/ml, IgG1, Serotec) or a combination of both mAbs, or mouse IgG1 as control [18,19]. After irradiation, the DCs derived in the absence or presence of IFN-β (100 U/ml) were incubated with the indicated mAbs for 30 min at 37°C. CD80 and CD86 was almost undetectable on the DCs that had been incubated with mAbs against CD80 or CD86 at concentration of 10 µg/ml when reanalysed by flow cytometry (data not shown). Then, MNCs were added and cultured for an additional 72 h. The supernatants were harvested for measurements of IL-2, IL-4 and IFN-γ production by ELISA. The cultures were pulsed with 1 µCi of [3H] thymidine. After 18 h, [3H] thymidine uptake was determined by scintillation counting.

Statistics

For group comparisons, the nonparametric Mann–Whitney test was used. For multiple comparisons, parametric anova test was used due to homogeneity of variances, followed by the Bonferroni test. For analysis of associations between cytokine levels, correlation and regression analysis was performed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

IFN-β and IL-10 suppress the immunostimulatory capacity of DCs in allogeneic mixed lymphocyte reactions

After 7 days' culture in GM-CSF and IL-4 without or with IFN-β at different concentrations (10–104 U/ml), DCs were tested for their stimulatory capacity to induce MNC proliferation in allogeneic MLR, that constitutes a specific function of DCs [1,3]. While IFN-β at lower concentrations (10 and 100 U/ml) had no effect (Fig. 2a), IFN-β at higher concentrations (1000 and 10 000 U/ml) inhibited the allostimulatory capacity of DCs (P < 0·05) (Fig. 2a). The concentration of 1000 U/ml of IFN-β per ml was chosen for the most experiments due to its significant influence on the allostimulatory capacity of DCs. The concentration of 1000 U/ml of IFN-β is 5–10 fold higher than the concentration achieved in the circulation by current treatment of MS with IFN-β-1b [20]. The concentration of 1000 U/ml has, however, been shown to inhibit IL-12 production by DCs [15] and to modulate functionally important molecules on DCs [21], and is widely used for in vitro studies [22].

image

Figure 2. Allogeneic mixed lymphocyte reactions (MLR) elicited by DCs from both MS patients (MS) and healthy controls (HC) are inhibited by IL-10 and IFN-β. Blood-derived DCs were cultured for 7 days with GM-CSF + IL-4, and subsequently exposed for 48 h to complete medium alone (control DCs □), TNF-α (50 ng/ml ), IL-10 (50 ng/ml ), IFN-β (1000 U/ml ) or IFN-β + IL-10 (▪). 1 × 104 irradiated DCs as stimulators were cocultured in 96-well round-bottomed microtitre plates with 2 × 105 allogeneic MNCs from healthy donors as responders. After 72 h, thymidine incorporation was measured by a 18-h pulse with [3H ]thymidine. (a) Effects of IFN-β at different concentrations on allostimulatory capacity of DCs in MLR. IFN-β at higher concentrations (1000 and 10 000 U/ml) inhibited the allostimulatory capacity of DCs in MLR (P < 0·05). IFN-β at lower concentrations (10 and 100 U/ml) had no significant effect on the allostimulatory capacity. Thymidine incorporation of MNCs alone was 3510 ± 562 cpm. The data are represented as mean ± SD of five independent experiments. (b) Proliferation of allogeneic MNCs in MLR is suppressed by DCs pretreated with IL-10 and IFN-β alone or IFN-β + IL-10, but enhanced by DCs pretreated with TNF-α(P < 0·01 for all comparisons) in both MS patients and HC. The values represent the mean ± SD from 20 healthy controls and 20 MS patients.

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DCs from both MS patients and healthy controls showed strong allostimulatory capacity in allogeneic MLR (Fig. 2b). IFN-β (1000 U/ml) as well as IL-10 (50 ng/ml) suppressed the allostimulatory capacity of DCs, while TNF-α (50 ng/ml) enhanced it (P < 0·01 for all comparisons). IFN-β combined with IL-10 also suppressed the allostimulatory capacity of DCs, but had no additive effect.

There were no differences between MS patients and healthy controls with respect to the allostimulatory capacity of blood DCs in MLR, irrespective of the absence or presence of IFN-β and IL-10. Nor were any differences found upon subgrouping the MS patients regarding treatment with IFN-β (data not shown). Thus, DCs from MS patients and healthy controls had similar allostimulatory capacity in MLR.

Augmented IFN production elicited by DCs derived from MS patients is inhibited by IFN-β and IL-10

To assess whether DCs from MS patients and healthy controls differ in their ability to induce production of Th1 cytokines by allogeneic MNCs in MLR, DCs from MS patients and healthy controls were irradiated and then cocultured with MNCs from healthy donors for 48 h. Culture supernatants were collected and analysed for the production of IFN-γ, IL-2 and IL-4 by ELISA.

As shown in Table 1, DCs from MS patients induced higher IFN-γ production by MNCs compared to DCs from healthy controls (1960 ± 1303 pg/ml versus 578 ± 457 pg/ml; P < 0·05). The production of IL-2 and IL-4 induced by DCs did not differ in MS patients and controls (Table 1). This indicates that DCs from MS patients favour Th1 cytokine production compared to DCs from healthy controls.

Table 1.  DC-elicited IFN-γ, IL-2 and IL-4 production (pg/ml) by allogeneic MNCs are modulated by IFN-β and IL-10.
 IFN-γIL-2IL-4
 MSHCMSHCMSHC
  1.   Results are presented as mean values ± SD.

  2.   P < 0.05 and *P <0.01 refer to comparisons of results obtained from DCs treated with the indicated cytokine versus control DCs (without cytokine treatment).

  3.   ND, not done.

Control DC1960 ± 1303578 ± 457418 ± 164407 ± 155170 ± 120200 ± 110
+IFN-β1298 ± 1030314 ± 286432 ± 219480 ± 235180 ± 110300 ± 300
+ IL-101127 ± 635390 ± 291326 ± 154359 ± 178NDND
+IFN+ IL-10902 ± 617*274 ± 231*295 ± 114305 ± 166NDND

DCs from MS patients and healthy controls, upon pretreatment with IL-10 (50 ng/ml) or IFN-β (1000 U/ml) alone only marginally reduced IFN-γ production by MNCs (Table 1). In contrast, pretreatment of DCs with IFN-β + IL-10 from both groups resulted in significant reduction of IFN-γ production by MNCs (P < 0·05). DCs pretreated with IFN-β or IL-10, or with IFN-β + IL-10 in combination had no significant effect on IL-2 production by MNCs in MS patients, nor in healthy controls. DCs pretreated with IFN-β had no significant effect on IL-2 production in both groups. Thus, DCs pretreated with IFN-β + IL-10 suppress Th1 responses.

We also examined production of IL-12 by DCs. IL-12 is a key cytokine to provide Th1 polarizing signal [5,23]. The production of IL-12 by DCs did not differ in MS patients and controls (Fig. 3). IL-12 production of DCs was inhibited by IFN-β (P < 0·05) or IL-10 (P < 0·05) in both groups. IFN-β + IL-10 displayed an additive inhibitory effect on IL-12 production by DCs in both MS patients and healthy controls (P < 0·01 for both). Thus DCs from MS patients and healthy controls showed similar capacity to produce IL-12.

image

Figure 3. IL-12 production by DCs is inhibited by IFN-β and IL-10. DCs were generated from blood adherent MNCs, cultured for 7 days in GM-CSF + IL-4, resuspended and incubated in the absence (□ control) or presence of IL-10(bsl00022), IFN-β (bsl00020) or IFN-β + IL-10 (▪) for an additional 48 h. The supernatants were collected and analysed for IL-12p40 production by ELISA. IFN-β and IL-10 alone had significant inhibitory effects on IL-12 production of DCs. IFN-β + IL-10 displayed an additive inhibition on IL-12 production of DCs derived from MS patients and healthy controls.

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Upon subgrouping the MS patients based on treatment with IFN-β, no differences were found for DC-elicited production of IFN-γ, IL-2 or IL-4 by MNCs or for IL-12 production by DCs. There were no correlation between production of any of these cytokines in the different groups.

DCs from MS patients show low expression of CD86 that is upregulated by IFN-β

Since the costimulatory pathway can influence Th polarization [24], we examined expression of CD80, CD86 and HLA-DR on DCs. The frequency of CD86+ DCs was lower in MS patients compared to healthy controls (25 ± 11% versus 39 ± 15%; P = 0·02) (Fig. 4). The density of CD86 expressed by per cell, examined as mean channel fluorescence (MCF), was 984 ± 440 in MS and 1024 ± 590 in healthy controls, revealing no statistically significant difference between the two groups. There were also no significant differences between IFN-β treated and untreated MS patients. The frequencies (%) and MCF of CD80+ and HLA-DR+ DCs did not differ between MS patients and healthy controls. Nor did MCF of CD80+ and HLA-DR+ DCs differ between the two groups (data not shown).

image

Figure 4. Percentages of CD80, CD86 and HLA-DR expressing DCs from MS patients and healthy controls (HC) are modulated by IL-10 (bsl00022), IFN-β (bsl00020) or IFN-β + IL-10 (▪). Compared to control DCs (□). DCs generated as shown in Fig. 3 were analysed by flow cytometry. (a) The level of CD80 on DCs from MS patients and HC is downregulated by IL-10 (P < 0·05), and upregulated by IFN-β (P < 0·05). (b) The baseline level of CD86 on DCs is lower in MS patients than in HC (P < 0·05). In both groups, IFN-β upregulates percentages of CD86 positive DCs (P < 0·05 in MS; P < 0·01 in HC). (c) The level of HLA-DR on DCs is downregulated by IL-10 and by IFN-β alone as well as by IL-10 + IFN-β in both MS and HC (P < 0·05). The values represent mean ± SD.

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Both in MS patients and healthy controls, in vitro exposure to IL-10 (50 ng/ml) reduced the percentages of CD80, CD86 and HLA-DR expressing DCs (P < 0·05 for all comparisons) (Fig. 4). In both MS patients and healthy controls, IFN-β (1000 U/ml) reduced the percentages of HLA-DR expressing DCs (P < 0·05 for both comparisons), but augmented the percentages of CD80 (P < 0·05 for both comparisons) as well as of CD86 expression (P < 0·05 in MS; P < 0·01 in healthy controls) (Fig. 4). DCs exposed to IFN-β + IL-10 also showed increased expression of CD80 and of CD86 in both groups, but to a less extent compared to IFN-β alone.

There were no differences for expression of CD80, CD86 or HLA-DR in response to IFN-β, IL-10 or IFN-β + IL-10 upon subgrouping the MS patients regarding treatment with IFN-β.

Anti-CD80 and anti-CD86 differentially influence DC-induced IL-4 production by MNCs

The role of costimulatory molecules CD80 and CD86 on the capacity of DCs to elicit Th1 and Th2 responses was examined. In three independent experiments performed on DCs from six healthy donors, anti-CD80 mAb (10 µg/ml), anti-CD86 mAb (10 µg/ml) and a combination of both mAbs was added to DC cultures in the absence or presence of IFN-β (100 U/ml) on day 7. Mouse IgG1 was used as control. MNCs were then added and cocultured with DCs for 72 h.

In all the cases, the costimulatory capacity of DCs in MLR was inhibited by anti-CD80 mAb, anti-CD86 mAb and the combination of both mAbs irrespective of the presence of IFN-β in the DC cultures (Fig. 5a). Correspondingly, DC-induced IL-2 (Fig. 5b) and IFN-γ(Fig. 5c) production by MNCs was suppressed by anti-CD80 and anti-CD86 mAb alone as well as in combination, regardless of the presence of IFN-β in the DC cultures. In agreement with another study [19], anti-CD80 mAb increased DC-induced IL-4 production, whereas anti-CD86 inhibited it (Fig. 5d) irrespective of presence of IFN-β in the DC cultures.

image

Figure 5. Effects of anti-CD80 and anti-CD86 mAbs on DC functions in polarizing Th responses in MLR. DCs (1 × 104) were generated in the absence or presence of IFN-β as shown in Fig. 3 and cultured together with 2 × 105 MNCs for 72 h in the presence of anti-CD80 (, 10 µg/ml, IgG1), anti-CD86 (, 10 µg/ml, IgG1), anti-CD80 and anti-CD86 in combination (▪) or mouse IgG1 (, control DCs). □ culture of MNCs alone. The supernatants were harvested and tested for IL-2, IL-4 and IFN-γ production by ELISA. The cells were pulsed with 1 µCi of [3H] thymidine. After 18 h, [3H] thymidine uptake was determined by scintillation counting. Co-stimulatory capacity of DCs generated without or with IFN-β (100 U/ml) in MLR (a) was inhibited by anti-CD80, anti-CD86 mAb, and a combination of both mAbs. DC-induced IL-2 (b) as well as IFN-γ (c) production by MNCs was suppressed by anti-CD80 and anti-CD86 mAb alone, and in combination, irrespective of IFN-β in the DC cultures. In contrast, (d) anti-CD80 mAb increased DC-induced IL-4 production, whereas anti-CD86 decreased it. Mean values and SD are shown. Results are from three independent experiments with MNCs from 3, and with DC from 6 healthy donors.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this study, we report that blood-derived DCs from MS patients compared to healthy controls showed augmented capacity to induce IFN-γ production by MNCs in MLR. In addition, the proportion of CD86+ DCs was low in MS patients. Upon exposure of DCs to IFN-β and IL-10 in vitro, IL-12 production by DCs was remarkably inhibited. Correspondingly, DC-induced IFN-γ production by MNCs was suppressed. CD86+ DCs were increased by IFN-β.

In active MS brain lesions, the inflammatory infiltrates consist of T cells, macrophages and other inflammatory cells [25], producing cytokines [26]. There is an increasing body of evidence that implies an imbalance between pro-inflammatory Th1 and anti-inflammatory Th2 cytokines in MS. The administration of Th1 cytokine IFN-γ induced MS exacerbation [27]. Transient increase in MS symptoms was associated with elevated levels of IFN-γ and TNF-α[28]. Decreased IL-10 and increased IL-12 p40 mRNA are associated with disease activity [29]. IFN-β treatment influences cytokine levels with increasing Th2 cytokine production [30,31] and decreasing proinflammatory Th1 cytokine production [32].

Efficient antigen uptake and abundant expression of MHC and costimulatory molecules [33,34] make DCs unique and critical as APCs in priming naive and memory T cells. We hypothesized that DCs from MS patients carry unknown signals, which promote Th1 immune response. To address this question, DCs were irradiated and cocultured with MNCs. Cytokines in relation to Th1 and Th2 responses were examined in the supernatants of the cultures. DCs derived from MS patients exhibited a stronger capacity to elicit IFN-γ production by MNCs than DCs derived from healthy controls. DC-induced IL-2 and IL-4 production by MNCs did not differ in MS patients versus healthy controls. This observation implies that DCs, under the conditions occurring in MS, favour Th1 cell responses.

No difference of IL-12 production by DCs was observed in MS patients versus healthy controls. IL-12 is an important inducer of IFN-γ production leading to Th1-biased response [35]. The observation does not exclude the possibility of IL-12 involvement in MS by its interaction with IL-12 receptors on effector cells such as T cell and by the responsiveness of immune cells to endogenous IL-12. The data show that cytokine requirements are complex and vary according to the circumstances.

Decreased CD86 expression found on DCs from MS patients could provide an explanation for the overproduction of DC-induced IFN-γ in allogeneic MNCs in MS. CD80 and CD86 molecules have different effects on Th cell differentiation. In some experimental systems, CD80 promotes a Th1 cell-mediated inflammatory response, CD86 elicits Th2 humoral response [24,36]. Circulating CD80+ B cells are increased during MS exacerbations, and the CD80/CD86 ratio on B cells and monocytes is increased during active MS [37]. During IFN-β therapy, the CD80/CD86 ratio approximates control levels. But CD86 expression blood lymphocytes are lower from relapsing MS patients, and CD80 expression is also increased per cell [38]. Increased expression of CD80 together with IL-12 p40 was also found in the acute MS plaques in the brain compared to what was found in the vascular infarcts in the brain associated with inflammation [39]. CD86, but not CD80, seems also to be essential for induction of low-dose oral tolerance [40], implicating distinct roles of the CD80 and CD86 molecules in modulating autoimmune reponses.

To investigate whether CD80 and CD86 are involved in Th1/Th2 polarization, mAbs against CD80 and CD86 were added to cultures of DCs [18,19]. Anti-CD80 and anti-CD86 alone and in combination abrogated costimulatory capacity of DCs in MLR. Correspondingly, DC-induced IL-2 production as well as IFN-γ production was suppressed in MLR (Fig. 5). These results support that the second ‘costimulatory’ signal delivered via CD80 or CD86 is essential in preventing Th cell inactivation and tolerance [41]. In our hands, anti-CD80 and anti-CD86 mAbs treatment showed different effects on DC-induced IL-4 production by MNCs, with anti-CD80 increasing DC-induced IL-4 production by MNCs and anti-CD86 decreasing it. The data are consistent with those of previous studies, showing that CD86 skews Th2 responses [24,36], but under certain circumstances. When both CD80 and CD86 molecules were blocked, DC-induced IL-4 production was not influenced. Overall, CD80 and CD86 signals are crucial for T cell responses. Th1/Th2 polarization seems not to be an intrinsic property of either CD80 or CD86. Rather, an imbalance between CD80 and CD86 may influence biased Th cell responses in MS.

We also investigated whether IFN-β affects the altered phenotype and function of DCs observed in MS, in comparison with IL-10, which inhibits DC maturation and activation [42,43]. The immunomodulatory activities of IFN-β may be more relevant to its effects in MS [15,21,44]. As immunomodulators, IFN-β possesses both anti- and pro-inflammatory activities [15]. The anti-inflammatory activity of IFN-β is beneficial in MS. But the pro-inflammatory activity may attenuate the beneficial effects in MS. Finding ways to inhibit its pro-inflammatory activity may augment the efficacy of IFN-β therapy. We selected IL-10 to outweigh pro-inflammatory activities of IFN-β. When DCs were pretreated with IL-10 + IFN-β, this combination had an additive inhibitory effect on IL-12 production compared to IL-10 or IFN-β alone. This effect was observed both in MS patients and healthy controls. Correspondingly, IFN-γ production by MNCs was significantly reduced by DCs pretreated with IL-10 + IFN-β. Together with the results by Wang et al. [22] that endogenous production of IL-10 is a required cofactor for the IFN-β-1b inhibitory effect on IL-12, the present findings suggest that effects obtained by IFN-β and IL-10 in combination on DCs in vitro could provide a novel mechanism to manipulate DCs in a desirable way in MS.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Julia Wilson and Robert Wallin (Microbiology and Tumour Biology Centre, Karolinska Institute, Stockholm, Sweden) for technical support. This study was supported by grants from the Swedish Medical Research Council and Karolinska Institute.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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