Serum IL-10 from systemic lupus erythematosus patients suppresses the differentiation and function of monocyte-derived dendritic cells

Abstract

The role played by cytokines, other than interferon (IFN)-α, in the differentiation and function of dendritic cells (DCs) in systemic lupus erythematosus (SLE), remains unclear. Serum interleukin-10 (IL-10) levels are generally elevated in SLE patients, which might modulate the differentiation of DCs. In this study, DCs were induced from monocytes either by transendothelial trafficking or by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) + IL-4 + tumor necrosis factor (TNF)-α. Both systems were used to investigate the effects of elevated serum IL-10 level on DC differentiation in SLE patients. The results showed that monocyte-derived DCs induced by either SLE serum or exogenous IL-10 reduced the expression of human leukocyte antigen (HLA)-DR and CD80, decreased IL-12p40 level, and increased IL-10 level, and exhibited an impaired capacity to stimulate allogenic T-cell proliferation. These results indicate that serum IL-10 may be involved in the pathogenesis of SLE by modulating the differentiation and function of DCs.

Keywords: dendritic cells (DCs); differentiation; interleukin-10 (IL-10); lupus erythematosus systemic (SLE).

Fig. 1. Effects of SLE serum on differentation of PBMCs.

A: Phenotypic comparison between PBMCs and non-migrated cells before and after incubation with the monolayer of human umbilical vein endothelial cells. PBMCs are incubated with the endothelial monolayer for 2 h before a thorough and careful wash to remove any non-migrated cells. Expression of CD11c, CD14, CD3, CD19, HLA-DR, CD80 and CD86 by the PBMCs before incubation, and by the non-migrated cells after incubation, are determined by flow cytometry. Results are expressed as mean±SD of percentage of the positive cells. The data are obtained from six independent experiments. *P < 0.05. B: Phenotypic analysis of the reverse-transmigrated cells cultured in the transendothelial. Expression of CD14, HLA-DR, CD80, CD86 and CD11c on the cell surface determined by flow cytometry. Cells are conjugated with fluorescent monoclonal antibodies. Re-sults of six separate experiments are expressed as mean±SD.

Fig. 2. Effects of SLE serum on functions of PBMCs.

A: Allostimulatory capacity of the reverse-transmigrated cells induced by the transendothelial trafficking model. Either the reverse-transmigrated cells induced by the transendothelial trafficking model, or the monocytes isolated from PBMCs, are used as stimulator cells and incubated with allogeneic CD3⁺ T cells at a ratio of 1:10. T-cell proliferation is measured by the [³H] TdR incorporation method. The degree of proliferation is indicated as the stimulation index (SI). Results are expressed as mean±SD. Data are obtained from six independent experiments. * P < 0.05. B: Effect of SLE serum on the allostimulatory capacity of MDDCs induced by the transendothelial trafficking model. MDDCs induced by the transendothelial trafficking model while culturing with different sera are used as stimulator cells and allogeneic CD3+ T cells are used as effec-tor cells. The two kinds of cells are incubated with each other at a ratio of 1:10. T-cell proliferation is measured by the [3H] TdR incorporation method. The degree of proliferation is indicated as the stimulation index (SI). Normal serum group, DCs are induced by the normal serum; SLE serum group, DCs are induced by the SLE serum in which IL-10 level is highly elevated (20-40 pg/mL) while the levels of other cytokines are not measured; IL-10 neutralized group, DCs are induced by the SLE serum plus anti-IL-10 neutralizing antibodies; Isotype control group, DCs are induced by the SLE serum plus rabbit anti-human IgG isotype controls; Exogenous IL-10 group, DCs are induced by the normal serum supplemented with exogenous IL-10 (30 pg/mL). Results are expressed as mean±SD. Data are obtained from six independent experiments. * P < 0.05.

Fig. 3. Effects of SLE serum on the phenotype of MDDCs induced by the transendothelial trafficking model.

MDDCs are induced by the transendothelial trafficking model and the expression of HLA-DR, CD80 and CD86 is determined by flow cytometry. Normal serum group, MDDCs are induced by the normal serum; SLE serum group, MDDCs are induced by the SLE serum with highly elevated levels of IL-10 (20-40 pg/mL) and normal levels of ILα and IL-6; IL-10 neutralized group, MDDCs are induced by the SLE serum plus anti-IL-10 neutralizing antibodies; Isotype control group, MDDCs are induced by the SLE serum plus rabbit anti-human IgG isotype controls; Exogenous IL-10 group, MDDCs are induced by the normal serum supplemented with exogenous IL-10 (30 pg/mL). A: The results of flow cytometry of different groups. B: The statistical results of flow cytometry. Results are expressed as mean±SD of the percentage of positive cells. Data are obtained from six independent experiments. *P< 0.05.

Fig. 4. Allostimulatory capacity of the cells induced by the GM-CSF + IL-4 + TNF-α culture system.

The cells induced by GM-CSF + IL-4 + TNF-α, or monocytes isolated from PBMCs, are used as stimulator cells and incubated with allogeneic CD3⁺ T cells at ratios of 1:10, 1:20, 1:50 and 1:100. T-cell proliferation is measured using cell counting kit-8. The degree of proliferation is indicated as the stimulation index (SI). Results are expressed as mean±SD. Data are obtained from five independent experiments. * P < 0.05.

Fig. 5. Effects of IL-10 on the phenotype of MDDCs induced by the GM-CSF + IL-4 + TNF-α culture system.

A: MDDCs were induced by GM-CSF + IL-4 + TNF-α with normal serum alone or with SLE serum containing different levels of IL-10. SLE serum group 1, SLE serum with normal levels of IL-10 (<10 pg/mL); SLE serum group 2, SLE serum with mildly elevated levels of IL-10 (10-20 pg/mL); SLE serum group 3, SLE serum with highly elevated levels of IL-10 (20-40 pg/mL). B: MDDCs are induced by the GM-CSF + IL-4 + TNF-α culture system with SLE serum containing highly elevated levels of IL-10 (20-40 pg/mL) or SLE serum plus anti-human IL-10 neutralizing antibodies. SLE serum plus rabbit anti-human IgG is served as isotype controls. C: MDDCs are induced by GM-CSF + IL-4 + TNF-α with or without exogenous IL-10 (30 pg/mL). The expression of HLA-DR, CD80 and CD86 is determined by flow cytometry. Results are expressed as mean±SD of the percentage of positive cells. Data are obtained from six independent experiments. * P < 0.05.

Fig. 6. Effects of IL-10 on the cytokine production by MDDCs induced by the GM-CSF + IL-4 + TNF-α culture system.

A: MDDCs are induced by GM-CSF + IL-4 + TNF-α with normal serum, SLE serum containing highly elevated levels IL-10 (20-40 pg/mL), SLE serum plus anti-human IL-10 neutralizing antibodies or SLE serum plus rabbit anti-human IgG isotype controls. B: MDDCs are induced by GM-CSF + IL-4 + TNF-α with or without exogenous IL-10 (30 pg/mL). The IL-12p40 and IL-10 concentrations in the culture supernatants of DCs are measured using ELISA kits. Results are expressed as mean±SD. Data are obtained from six independent experiments. * P < 0.05.

Fig. 7. Effects of IL-10 on the allostimulatory capacity of MDDCs induced by the GM-CSF + IL-4 + TNF-α culture system.

A: MDDCs are induced by GM-CSF + IL-4 + TNF-α with normal serum, SLE serum containing highly elevated levels IL-10 (20-40 pg/mL), SLE serum plus anti-human IL-10 neutralizing antibodies or SLE serum plus rabbit anti-human IgG isotype controls. B: MDDCs are induced by GM-CSF + IL-4 + TNF-α with or without exogenous IL-10 (30 pg/mL). DCs are then used as stimulator cells and incubated with allogeneic CD3⁺ T cells at DC:T cell ratios of 1:10, 1:20, 1:50 and 1:100. T-cell proliferation is measured using cell counting kit-8. The degree of proliferation is indicated as the stimulation index (SI). Results are expressed as mean±SD. Data are obtained from six independent experiments. * P < 0.05.