Immunomodulatory effects of cyclosporin A on human peripheral blood dendritic cell subsets

Abstract

Cyclosporin A (CsA) is a potent immuno-suppressant and is approved for the treatment of various disease conditions. The molecular biological mechanism of CsA has been investigated intensively in T cells and has been shown to involve the intracellular calcineurin pathway. Recently, it was reported that CsA has capacities to affect not only T cells but also antigen-presenting cells such as B cells and dendritic cells (DCs). DCs are a master regulator of immune responses that have an integral capacity to prime naive T cells. In the present study, we investigated the biological effects of CsA on human peripheral blood DC subsets: CD11c+ myeloid and CD11c- lymphoid subsets. CsA inhibited the up-regulation of co-stimulatory molecules induced with or without microbial stimuli and CD40L on both CD11c+ and CD11c- subsets. In addition, CsA negatively regulated the endocytic activity of CD11c+ DC during the immature state. CsA inhibited the interleukin-12 (IL-12) production, but augmented the IL-10 production from the LPS-stimulated CD11c+ subset, whereas CsA reduced the interferon-alpha (IFN-alpha) production from the CD11c- subset infected with Sendai virus (SV). Both the LPS-stimulated CD11c+ subset and SV-infected CD11c- subset preferentially induced the development of IFN-gamma-producing T helper-type 1 (Th1) cells. Pretreatment of these DC subsets with CsA inhibited the Th1 skewing. These findings suggested a DC-mediated mechanism of immunosuppression by CsA.

Figure 1

Effect of CsA on viability of DC. CD11c⁺ and CD11c⁻ DC subsets were cultured with medium alone and IL-3, respectively. Both DC subsets were incubated with different concentrations of CsA for 3 days. The number of viable cells in each DC subset was evaluated as annexin V-negative fractions by flow cytometry. The data represent the means ± SEM of four independent experiments.

Figure 2

Effect of CsA on maturation of DCs. CD11c⁺ and CD11c⁻ DC subsets were cultured with medium alone and IL-3, respectively. Then, CD11c⁺ and CD11c⁻ DC subsets were cultured with different stimuli indicated in the presence or absence of CsA for 72 hr. The expression levels of CD40, CD80, CD86 and HLA-DQ on each DC subset were analysed by flow cytometry. Results were shown as ΔMFI, which is calculated by subtraction of MFI with the isotype-matched control from that with each mAb. The results shown here are representative of four independent experiments.

Figure 3

Effect of CsA on MMR expression and endocytic activity of CD11c⁺DCs. CD11c⁺ DC subset was cultured with medium alone in the presence of different concentrations of CsA for 24 hr. (a) Expression of MMR was measured before and after the culture. Percentage of positive cells was indicated in the figure. (b) After incubation with FITC-dextran at 37° for 30 or 60 min, dextran uptake was analysed by flow cytometry. The results are shown as MFI, from which the background fluorescence (incubated at 4°) is subtracted. The results shown in this figure are representative of four independent experiments.

Figure 4

Effect of CsA on allostimulatory capacity of DCs. CD11c⁺ DCs were cultured with GM-CSF + sCD40L or LPS in the presence or absence of CsA (500 ng/ml), and CD11c⁻ DCs were cultured with IL-3 + sCD40L or SV in the presence or absence of CsA (500 ng/ml). Graded doses of the cultured DCs (stimulator cells) were irradiated and co-cultured with allogeneic naive CD4⁺ T cells. T cell proliferation was measured by BrdU incorporation. The results are shown as means of triplicate cultures. Vertical bars represent SEM of triplicate cultures (*P < 0·05). Representative data of three experiments are shown.

Figure 5

Effect of CsA on cytokine production of DCs.After 24 hr-culture with LPS or SV in the presence or absence of CsA (500 ng/ml), supernatants of DCs were measured for IL-12 p40 + 70, IL-12 p70, IL-10 and IFN-α by ELISA. Statistical significance is indicated using paired Student's t-test (*P < 0·05). The data represent the means ± SEM of three independent experiments.

Figure 6

Effect of CsA on DC-mediated Th polarization. (a,b) CD11c⁺ and CD11c⁻ DCs were preincubated with LPS and SV, respectively, in the presence or the absence of CsA (500 ng/ml). These DCs were washed extensively and then co-cultured with allogeneic naive CD4⁺ T cells for 7 days. The T cells were restimulated with PMA and ionomycin for 6 hr. Brefeldin A was added to the cultures for the last 2 hr, and then intracellular IFN-γ, IL-4 and IL-10 of T cells was analysed by flow cytometry. The percentages of the respective cytokine-producing T cells are indicated in each dot blot profile. (c) Percentages of IFN-γ-producing Th cells are shown (upper; CD11c⁺ DCs, lower; CD11c⁻ DCs). Data represent the means ± SEM of five independent experiments. Statistical significance is indicated using paired Student's t-test (*P < 0·05). Data are presented as means ± SEM.