Tumour necrosis factor-α but not lipopolysaccharide enhances preference of murine dendritic cells for Th2 differentiation

Abstract

Using murine spleen-derived dendritic cells (DC) and DO11.10 T cells specific for ovalbumin (OVA), the influences of maturational condition and antigen dose on the capability of DC to induce helper T-cell (Th) differentiation were analysed. Immature DC (iDC) with high- or low-dose OVA_323–339 predominantly induced Th1 or Th2 responses in DO11.10 T cells, respectively. DC matured by tumour necrosis factor-α (TNF/DC) induced a significantly higher Th2 response in the presence of low-dose OVA_323–339 than iDC and DC matured by lipopolysaccharide (LPS) (LPS/DC). In the presence of high-dose OVA_323–339, LPS/DC induced significantly lower levels of Th1 response than iDC. Under these conditions no difference in the Th1 response was noted between TNF/DC and iDC. The enhanced capability of TNF/DC with a low-dose antigen for Th2 polarization and the decreased preference of LPS/DC with a high-dose antigen to Th1 polarization were not related to the amount of IL-12 produced in these cultures. These results demonstrate for the first time that TNF/DC with a low-dose antigen are potent inducers of Th2 differentiation.

Introduction

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) and play major roles in the initiation and regulation of adaptive immune responses to antigens.^1–3 Upon encountering foreign antigens, DC are rapidly activated by a complex process and become mature.^2–4 DC activation in the inflammatory tissues is triggered by cytokines, including tumour necrosis factor-α (TNF-α), and bacterial components, including lipopolysaccharide (LPS).^2–6 The mature DC highly express major histocompatibility complex (MHC) and co-stimulatory molecules, including CD80, CD86 and CD40, on their surface. The mature DC migrate from peripheral tissues to the T-cell area of draining lymph nodes where the DC activate antigen-specific T cells.^2,3

It has been shown in the murine system that two types of DC are present, which express either high levels of CD8α or CD11b molecules. CD8⁺ or CD11b⁺ DC predominantly promote T helper type 1 (Th1) or type 2 (Th2) responses, respectively.^7–9 In addition to the type of DC, the Th polarization appears to be regulated by microenvironmental conditions during antigen presentation.¹⁰ It has been reported that variable factors are involved in the Th polarization; genetic predisposition,^11,12 cytokine gene chromatin structure,¹³ cell cycle,¹⁴ strength of co-stimulation provided by APC,^15–17 avidity of T-cell receptor (TCR)–ligand interaction,^15,18 DC : T-cell ratio,¹⁹ antigen dose,^20–22 and antigen type.^23–25 Thus, the physiological mechanism underlying Th polarization appears to be extremely complex and the elucidation of the exact mechanism is far from complete.

TNF-α is highly expressed in inflammatory sites and promotes DC maturation, such that TNF-α markedly increases surface expression of MHC and co-stimulatory molecules on DC.^{2,3,5,26–28} In our previous study, we established a murine spleen-derived DC line (BC1)²⁸ and showed that TNF-α enhanced the allostimulatory capability of murine DC for T-cell proliferation. However, the effect of TNF-α on the DC capability to promote Th1 or Th2 differentiation has been unclear. In the present study, using the BC1 cells, we evaluated the effects of TNF-α and LPS on the capability of DC for Th differentiation. We demonstrate herein that TNF-α but not LPS enhances the preference of DC for Th2 differentiation.

Materials and methods

Animals

BALB/c mice were purchased from Japan SLC Inc. (Hamamatsu, Shizuoka, Japan). Ovalbumin (OVA)-specific TCR-transgenic mice (DO11.10 mice)²⁹ were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and were maintained in specific pathogen-free conditions at our animal facility at Hokkaido University. Mice were used at 6–12 weeks of age. All experiments were approved by the regulations of Hokkaido University Animal Care and Use Committee.

Reagents and antibodies

Recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF) and TNF-α were purchased from PeproTech (London, UK). OVA_323–339 peptide (ISQAVHAAHAEINEAGR) was synthesized using an automatic peptide synthesizer PSSM-8 (Shimadzu, Kyoto, Japan) and purified by reverse-phase high-performance liquid chromatography (Waters Japan, Tokyo, Japan) as previously described.³⁰ Fluorescein isothiocyanate (FITC)-conjugated anti-mouse interferon-γ (IFN-γ) monoclonal antibody (mAb) (XMG1.2), FITC-conjugated anti-mouse CD86 mAb (GL1), FITC-conjugated anti-mouse CD40 mAb (3/23), Phycoerythrin (PE)-conjugated anti-mouse CD11c mAb (HL3), PE-conjugated anti-mouse CD80 mAb (16-10A1), biotin-conjugated anti-mouse H-2K^d mAb (SF1-1.1), biotin-conjugated anti-mouse H-2A^d mAb (AMS-32.1), and streptavidin Cy-Chrome™ were obtained from PharMingen (La Jolla, CA). PE-conjugated anti-mouse interleukin-4 (IL-4) mAb (BVD4-1D11) was purchased from Caltag Laboratories (Burlingame, CA). As control immunoglobulin (IgG), FITC-conjugated rat IgG2a, FITC-conjugated rat IgG1, and PE-conjugated rat IgG2b were purchased from PharMingen. Biotin-conjugated mouse IgG2b was purchased from Immunotech (Marseille, France). Biotin-conjugated mouse IgG2a was purchased from Dako (Copenhagen, Denmark). PE-conjugated hamster IgG was obtained from Caltag Laboratories. A clonotypic mAb for the transgenic TCR of DO11.10 mice, KJ1-26,²⁹ was purified from hybridoma supernatant with Hi-Trap Protein G (Amersham Pharmacia Biotech, Uppsala, Sweden) and biotinylated by incubating with N-hydroxy-succimide biotin (Pierce, Rockford, IL) in dimethyl sulphoxide solution.³¹

Culture media

The culture media used were Iscove's modified Dulbecco's medium (IMDM) and RPMI-1640 (Sigma Chemical Co., St Louis, MO) supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 600 μg/ml l-glutamine, and 50 μm 2-mercaptoethanol (complete IMDM and complete RPMI-1640, respectively). Fibroblast supernatants from NIH/3T3 cells were collected from confluent cultures with complete IMDM.²⁸

Spleen-derived DC line (BC1)

A spleen-derived dendritic cell line (BC1) from BALB/c mice was generated as described in our previous report.²⁸ BC1 cells are CD11c⁺, MHC class I⁺, MHC class II⁺, CD80⁺, and CD86⁺.²⁸ Although BC1 cells show an immature phenotype, various activating stimuli, such as TNF-α and LPS, promote maturation of this precursor line. The mature BC1 cells highly express MHC and co-stimulatory molecules and have a potent allostimulatory capability as compared with immature cells.²⁸ BC1 cells were expanded in complete IMDM containing 30% NIH/3T3 supernatant and 10 ng/ml mouse recombinant GM-CSF (henceforth referred as R1 medium).^27,28,32 In the present study, BC1 cells (2 × 10⁵ cells/ml) pretreated with 5 μg/ml LPS or 40 ng/ml TNF-α for 24 hr were used as mature DC.

Purification of DO11.10 CD4⁺ T cells

Lymphocytes prepared from the lymph nodes of DO11.10 mice were incubated with RA3-6B2 (anti-mouse B220 mAb) and MKD6 (anti-mouse H-2A^d mAb) culture supernatant. The cells were washed three times and incubated with goat anti-rat IgG microbeads, goat anti-mouse IgG microbeads and anti-mouse CD8α mAb microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). CD4⁺ T cells were purified (> 97% purity) by depleting MHC class II⁺, B220⁺, and CD8α⁺ cells using the MACS system (Miltenyi Biotec).

T-cell proliferation assay

The T-cell proliferation assay was performed using DO11.10 CD4⁺ T cells as responder cells and BC1 cells as APC. BC1 cells were pretreated with or without TNF-α (40 ng/ml) or LPS (5 μg/ml) for 24 hr. Cells were detached, washed three times with complete RPMI-1640, and used as immature DC or mature DC. APC (2 × 10⁴ cells) and responder cells (4 × 10⁴ cells) were co-cultured in a 96-well culture plate with various concentrations of OVA_323–339 (0·001∼10 μm) in 280 μl of complete RPMI-1640 at 37° in 5% CO₂. After 48 hr, cultures were pulsed with 0·5 μCi/well of [³H]thymidine (Amersham, Tokyo, Japan) for 12 hr and then harvested onto glass fibre. Incorporation of [³H]thymidine was measured with a gas flow β counter (Matrix 96; Packard, Meriden, CT).

Flow cytometry

BC1 cells were incubated with 2.4G2 (rat anti-mouse FcγRII/III, CD32) culture supernatant to prevent binding to FcRII/III, and then stained using FITC-, PE-, or biotin-conjugated mAb and streptavidin-Cy-Chrome™.³¹ Flow cytometry was performed on an EPICS^® XL (Coulter Co., Miami, FL), as previously described.³³

In vitro differentiation of T cells and analysis of intracellular cytokine

Th polarization of DO11.10 CD4⁺ T cells was analysed as previously described.²² Immature or mature BC1 cells were prepared as described above. For the primary response, DO11.10 T cells (1 × 10⁵ cells) and BC1 cells (5 × 10⁴ cells) were co-cultured in a 48-well culture plate with various concentrations of OVA_323–339 (0·001∼10 μm) in 700 μl of complete RPMI-1640 at 37° in 5% CO₂. After 3 days newly prepared BC1 cells (5 × 10⁴ cells) were added for the secondary stimulation and cultures were continued in the presence of the same concentration of OVA_323–339 in 1·5 ml complete RPMI-1640 in a 24-well plate. Two days later, T cells were stimulated with phorbol 12-myristate 13-acetate (Sigma-Aldrich Co.) (50 ng/ml) and A23187 (Sigma-Aldrich Co.) (500 ng/ml) for 4 hr. Two hours prior to harvesting, monensin (1 μg/ml) was added. Cells were stained with KJ1-26 biotin-conjugated mAb and streptavidin-Cy-Chrome™ as a second reagent.²⁹ Then the cells were fixed with paraformaldehyde and permeabilized using 0·5% saponin. Finally, the cells were stained with FITC-labelled anti-IFN-γ and PE-labelled anti-IL-4. Flow cytometry was performed on an EPICS^® XL (Coulter Co.).

Measurement of IL-12 p70 and IL-12 p40 by enzyme-linked immunosorbent assay (ELISA)

After a T-cell proliferation assay for 1, 3, or 5 days, culture supernatants were subjected to quantification of the protein level of IL-12 p70 and IL-12 p40 by ELISA using commercially available mouse IL-12 p40 and IL-12 p70 immunoassay kits (BioSource International, Camarillo CA).

Statistical analysis

The Student's t-test was used to analyse data for significant differences. P-values less than 0·05 were regarded as significant.

Results

Phenotypical and functional maturation of BC1 cells induced by TNF-α or LPS

We have reported that unstimulated BC1 cells are phenotypically and functionally immature DC (iDC).²⁸ Figure 1 shows the effects of TNF-α and LPS on the expression of MHC and co-stimulatory molecules on the surface of BC1 cells. BC1 cells were cultured with TNF-α at 40 ng/ml or LPS at 5 μg/ml for 24 hr (TNF/DC or LPS/DC), and the expressions of MHC class II, MHC class I, CD86, CD80 and CD40 were analysed by flow cytometry. Either TNF-α or LPS significantly enhanced the expression of these molecules on BC1 cells (Fig. 1a). The enhancement of CD40, CD86 and CD80 expressions by LPS was statistically more prominent than that by TNF-α (Fig. 1b). On the other hand, the expressions of MHC class I and class II on TNF/DC fell within the levels similar to those on LPS/DC. Since the expressions of these molecules reached a peak at a concentration of 40 ng/ml of TNF-α or 5 μg/ml of LPS, respectively (data not shown), these doses were used to treat BC1 cells throughout this study.

Figure 1.

TNF-α and LPS-induced phenotypic maturation of BC1 cells. BC1 cells were treated with TNF-α (40 ng/ml) or LPS (5 μg/ml) for 24 hr. Expressions of MHC and co-stimulatory molecules were analysed by flow cytometry. (a) Representative histogram of five independent experiments. (b) Mean intensity (mean ± SE) of five independent experiments. Statistical significance was calculated by the Student's t-test (*P < 0·05, **P < 0·01).

Then, to examine the effect of TNF-α or LPS on the capability of BC1 cells to induce proliferation of antigen-specific CD4⁺ T cells, a T-cell proliferation assay was performed using CD4⁺ DO11.10 T cells as responder cells in the presence of various doses of OVA_323–339 peptide. As shown in Fig. 2, TNF/DC, LPS/DC and iDC generated antigen-specific T-cell proliferation in an antigen dose-dependent manner. It should be noted in Fig. 2 that LPS/DC were the most potent APC for the T-cell proliferation and TNF/DC were the second most potent APC.

Figure 2.

Capability of BC1 cells to induce antigen-specific T-cell proliferation. After treatment with TNF-α (40 ng/ml) or LPS (5 μg/ml) for 24 hr, BC1 cells were washed and irradiated (30 Gy). The cells (2 × 10⁴) were co-cultured with DO11.10 CD4⁺ T cells (4 × 10⁴) in the presence of variable concentrations of OVA_323–339 peptide (0·001∼10 μm). After 48 hr, the cultures were pulsed with [³H]thymidine for 12 hr and incorporated [³H]thymidine was counted. Each symbol represents mean ± SE of triplicate. Data are representative of three independent experiments.

Effect of antigen dose on Th1/Th2 polarization

To examine the role of antigen dose in Th1/Th2 polarization by iDC, DO11.10 CD4⁺ T cells were co-cultured with BC1 cells in the presence of various concentrations of OVA_323–339 peptide (0·01–10 μm). After 5 days of culture the Th1 and Th2 polarization was analysed by intracellular staining of IFN-γ and IL-4. As shown in Fig. 3(a), a direct relationship was detected between the antigen dose and Th1/Th2 polarization. In the presence of iDC the proportion of Th1 (IFN-γ⁺ IL-4^–) cells was increased in an antigen dose-dependent manner (Fig. 3b). On the other hand, the proportion of Th2 (IFN-γ^– IL-4⁺) cells and the antigen dose followed an inverse relationship. A very low frequency (<4%) of IFN-γ⁺ IL-4⁺ Th cells could be detected in every concentration of antigen (Fig. 3a).

Figure 3.

Effect of antigen dose on Th1/Th2 polarization. DO11.10 CD4⁺ T cells were co-cultured with BC1 cells in the presence of various concentrations of OVA_323–339 peptide. The cells were stained with KJ1-26 mAb and monitored for intracellular staining of IFN-γ and IL-4. (a) Dot plots gated by KJ1-26-positive cells. Results are representative of two independent experiments. (b) Proportions of IFN-γ⁺ IL-4^– or IL-4⁺ IFN-γ^– cells in the KJ1-26-positive cells. Mean of two independent experiments.

Effect of TNF-α and LPS on capability of BC1 cells for Th1/Th2 differentiation

To examine whether maturation stage of DC affects not only T-cell proliferation but also Th1/Th2 polarization, we analysed intracellular expressions of IFN-γ and IL-4 in DO11.10 (KJ1-26⁺) CD4⁺ T cells after co-culture with iDC, TNF/DC, or LPS/DC in the presence of a high or low dose of OVA_323–339 peptide for 5 days. In the absence of antigen, the proportion of IFN-γ⁺ or IL-4⁺ cells in the culture of any DC plus DO11.10 T cells was less than 1% (data not shown). In the culture with iDC, the proportion of Th1 (IFN-γ⁺ IL-4^–) cells was 9·3% or 56% in the presence of a low or high dose of OVA_323–339, respectively (Fig. 4a). On the other hand, the proportion of Th2 (IFN-γ^– IL-4⁺) cells was 15% or 1·1% in the presence of a low or high dose of OVA_323–339 peptide. These findings were principally consistent with prior results (Fig. 3). Similarly TNF/DC and LPS/DC with the high-dose antigen induced predominantly Th1 differentiation, whereas those with the low-dose antigen induced mainly Th2 polarization (Fig. 4a). In Fig. 4(b) the mean proportions ± SEM of Th2 and Th1 cells in each group (n = 10) are shown. The mean proportion of Th1 cells in the culture with TNF/DC or LPS/DC plus 10 μm OVA_323–339 was slightly or significantly lower than that in the culture with iDC plus 10 μm OVA_323–339, respectively (Fig. 4b, right panel). In the presence of a 0·01-μm OVA_323–339, TNF/DC predominantly promoted Th2 responses. The mean proportion of Th2 cells induced by TNF/DC with the low-dose antigen was significantly higher than that by iDC or LPS/DC. No difference was seen in the Th2 polarization between iDC and LPS/DC (Fig. 4b, left panel).

Figure 4.

Effect of TNF-α or LPS on preference of BC1 for Th differentiation. DO11.10 CD4⁺ T cells were co-cultured with unstimulated BC1 cells (iDC), BC1 cells matured by TNF-α or LPS (TNF/DC or LPS/DC) in the presence of a high- or low-dose OVA_323–339 peptide. The cells were stained by KJ1-26 mAb and monitored for intracellular staining of IFN-γ and IL-4. (a) Dot plots gated by KJ1-26 positive cells. Results are representative of 10 independent experiments. (b) Proportions of IFN-γ⁺ IL-4^– or IL-4⁺ IFN-γ^– cells in the KJ1-26 positive cells. Mean ± SE of 10 independent experiments. Statistical significance was calculated by the Student's t-test (**P < 0·01).

IL-12 production during the antigen presentation by iDC, TNF/DC, or LPS/DC with a high- or low-dose antigen

It has been reported that IL-12 is produced by DC during the antigen presentation.³⁴ Figure 5 shows IL-12 production in the culture of CD4⁺ DO11.10 T cells and iDC, TNF/DC, or LPS/DC with a low (0·01 μm) or high (10 μm) dose OVA_323–339 after 1, 3, or 5 days of culture. IL-12 p40 production in the culture with iDC plus 10 μm antigen was larger than that with iDC plus 0·01 μm antigen for the entire period examined (day 3, P < 0·05, day 5, P < 0·01) (Fig. 5a). Similarly, TNF/DC with the high-dose antigen produced significantly larger amounts of IL-12 p40 than those with the low-dose antigen at days 1, 3 and 5 (Fig. 5a). However, in the presence of 10 μm antigen, the amount of IL-12 p40 produced by TNF/DC was significantly smaller than that produced by iDC at days 3 and 5 (Fig. 5a). In contrast, IL-12 p40 production by LPS/DC with 0·01 or 10 μm antigen fell within almost the same level as at days 1, 3 and 5. It should be noted that in Fig. 5(a) the IL-12 p40 production by LPS/DC with either low- or high-dose antigen was significantly larger than that by iDC with the high-dose antigen at day 1. At days 3 and 5, levels of IL-12 p40 production by LPS/DC were similar to those by iDC with the high-dose antigen (Fig. 5a).

Figure 5.

IL-12 production during the antigen presentation by BC1 cells matured by TNF-α or LPS. CD4⁺ T cells were co-cultured with unstimulated BC1 cells (iDC) or BC1 cells matured by TNF-α or LPS (TNF/DC or LPS/DC) in the presence of a high- or low-dose OVA_323–339 peptide. After 1, 3, or 5 days, the supernatants were collected and the amounts of IL-12p40 and IL-12p70 were measured using an IL-12p40 or IL-12p70 ELISA kit. (a) Concentrations of IL-12 p40 in the culture supernatants. (b) Concentration of IL-12 p70 in the culture supernatants. Each symbol represents the mean ± SE of three (day 1 and 3) or nine (day 5) independent experiments. Statistical significance was calculated by the Student's t-test (*P < 0·05, **P < 0·01).

The production of IL-12 p70 showed almost the same pattern as that of IL-12 p40 at day 5, although the amount of IL-12 p70 was low compared to that of IL-12 p40 (Fig. 5b). Amounts of IL-12 p70 in the culture at days 1 and 3 were under the detectable level in any conditions (Fig. 5b).

Discussion

Activation and maturational states of DC are regulated by various extracellular stimuli including cytokines, co-stimulatory molecules and bacterial products. These events are closely related to alterations of the morphological, phenotypical and functional properties of DC.^1–5 TNF-α, an inflammatory cytokine, strongly promotes the activation and maturation of DC. These TNF-α-induced mature DC in the inflammatory site move to the T-cell area of draining lymph nodes and activate the antigen-specific T cells. Thus, the maturational step of DC by TNF-α is an important component for appropriate antigen presentation. However, the effect of TNF-α on DC functions, especially the capability to induce T-cell differentiation, is not fully understood.

In the present study using an immature DC cell line (BC1) we analysed the influences of TNF-α on maturation of DC and compared them with those of LPS. Using a similar culture system, a number of important findings have been reported and verified.^27,35–40 We showed herein that in the presence of a low-dose antigen TNF/DC induced preferentially Th2 responses in DO11.10 T cells compared to LPS/DC or iDC (Fig. 4).

Both TNF-α and LPS markedly increased the expressions of MHC and co-stimulatory molecules on iDC (Fig. 1). However, expressions of CD80, CD86 and CD40 were more markedly enhanced by LPS than by TNF-α. No significant difference in the expression of MHC class II was detected between LPS/DC and TNF/DC. The capability of LPS/DC for T-cell proliferation was more vigorous than that of TNF/DC. Thus, this high capability of LPS/DC to induce T-cell proliferation may be attributed to the high expressions of co-stimulatory molecules as compared with TNF/DC.

It has been reported that antigen dose affects the APC-induced Th1/Th2 polarization.^20–22 In agreement with these previous reports, all DC studied herein with the high- or low-dose antigens predominantly induced Th1 or Th2 responses, respectively (Figs 3, 4). During the presentation of high-dose antigens (day1∼5), iDC produced large amounts of IL-12 compared to those produced in the presence of low-dose antigens (Fig. 5). Thus, the predominant Th1 differentiation induced by iDC plus high-dose antigens is presumably attributed to the vigorous production of IL-12 during the antigen presentation. Similarly IL-12 production by TNF/DC with the high-dose antigens was larger than that with low-dose antigens during the entire period examined (Fig. 5). However, when compared to iDC culture, IL-12 production in the culture of TNF/DC with high-dose antigens was significantly lower at day 3 and 5 (Fig. 5). Nevertheless, no considerable differences in the proportion of Th1-type cells were detected between the cultures with TNF/DC and those with iDC (Fig. 4). Thus, it seemed that the amount of IL-12 in the culture with TNF/DC plus high-dose antigens was sufficient to induce the full Th1 differentiation.

It should be noted that TNF/DC promoted Th2 differentiation more preferentially than iDC in the presence of a low-dose antigen. Almost the same levels of IL-12 were produced by TNF/DC and by iDC in these cultures at days 1∼5 (Fig. 5). Thus, the enhanced capability of TNF/DC plus low-dose antigens for Th2 polarization compared to that of iDC was unlikely to be related to the amount of IL-12 in these cultures. Components other than IL-12 appeared to contribute to the enhancement of TNF/DC capability for Th2 polarization. We would like to postulate that antigen presentation with a low-dose antigen and considerably high expression of co-stimulatory molecules may be related to the remarkable Th2 polarization by TNF/DC.

Similarly, in the presence of low-dose antigens, LPS/DC also seemed to express a low density of antigen–MHC complex with large amounts of co-stimulatory molecules. However, these LPS/DC induced significantly lower Th2 responses than TNF/DC (Fig. 4). It seems that the low preference of LPS/DC for Th2 differentiation is related to their vigorous production of IL-12 during the entire period of the culture (Fig. 5).

Recently, Langenkamp et al.²¹ reported that preference of human monocyte-derived DC for Th1 differentiation was reduced by LPS treatment for 48 hr. These authors observed that DC produced IL-12 during a narrow time window, 10–18 hr, after stimulation with LPS. Thereafter the DC became refractory to further stimulation and was unable to produce IL-12. These findings suggest that the decreased preference of LPS/DC for Th1 differentiation is attributed to insufficient IL-12 production during the antigen presentation. In the present study, we also demonstrated that preference of LPS/DC for Th1 differentiation was considerably low as compared with that of iDC in the presence of high-dose antigens (Fig. 4). However, in this condition, rather higher levels of IL-12 were produced by LPS/DC than by iDC at day 1, and almost the same levels of IL-12 were produced at days 3 and 5 (Fig. 5). These findings suggest that a mechanism other than the IL-12 level plays a role in the decreased preference of LPS/DC for Th1 differentiation, and that the pathway of LPS-induced IL-12 production may be different between murine spleen-derived DC and human monocyte-derived DC. Surface expressions of co-stimulatory molecules on LPS/DC were very high compared with those on TNF/DC (Fig. 1). We consider that the balance between TCR-mediated signals and those mediated through co-stimulatory molecules may influence the differentiation of Th cells. This possibility should be analysed in future investigations.

We showed herein a novel role of TNF-α in the regulation of DC preference for Th1/Th2 polarization. Since the Th1/Th2 balance is related to various immunological disorders, allergy, autoimmune disorders, etc., our present finding could be a basis from which to develop useful and powerful strategies that regulate these immune disorders in clinical applications.

Abbreviations

APC

antigen-presenting cells

mAb

monoclonal antibody

DC

dendritic cells

OVA

ovalbumin

TCR

T-cell receptor

Th

helper T cell

Th1

T helper cell type 1

Th2

T helper cell type 2

IFN-γ

interferon-γ

IL-4

interleukin-4

LPS

lipopolysaccharide

TNF-α

tumour necrosis factor-α