Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses - PubMed (original) (raw)

Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses

M Rescigno et al. J Exp Med. 2000.

Abstract

Ligation of the Fas (CD95) receptor leads to an apoptotic death signal in T cells, B cells, and macrophages. However, human CD34(+)-derived dendritic cells (DCs) and mouse DCs, regardless of their maturation state, are not susceptible to Fas-induced cell death. This resistance correlates with the constitutive expression of the Fas-associated death domain-like IL-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP) ligand. We demonstrate a new role of Fas in DC physiology. Engagement of Fas on immature DCs by Fas ligand (FasL) or by anti-Fas antibodies induces the phenotypical and functional maturation of primary DCs. Fas-activated DCs upregulate the expression of the major histocompatibility complex class II, B7, and DC-lysosome-associated membrane protein (DC-LAMP) molecules and secrete proinflammatory cytokines, in particular interleukin (IL)-1beta and tumor necrosis factor alpha. Mature DCs, if exposed to FasL, produce even higher amounts of IL-1beta. Importantly, it is possible to reduce the production of IL-1beta and interferon (IFN)-gamma during DC-T cell interaction by blocking the coupling of Fas-FasL with a Fas competitor. Finally, during cognate DC-T cell recognition, IL-12 (p70) could not be detected at early or late time points, indicating that Fas-induced, IFN-gamma secretion is independent of IL-12.

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Figures

Figure 1

Figure 1

Surface levels of Fas (CD95) in immature versus mature DCs. (A) Human CD34+–derived DCs were treated with 40 ng/ml of TNF-α for 24 h (solid line) or left untreated (dashed line); isotype control (gray line). Results are representative of three independent experiments. (B) Mouse D1 cells or (C) mBM-DCs were treated with 10 μg/ml LPS. Thick line, treated cells; thin line, isotype control; dashed line, untreated cells (NT). Results are representative of four experiments.

Figure 3

Figure 3

Fas engagement triggers the phenotypical maturation of human and mouse DCs. (A) Surface expression of cellular receptors and intracellular expression of DC-LAMP in human CD34+–derived DCs treated with 0.1 μg/ml FasL plus 0.5 μg/ml Enhancer for 24 h (black line) versus 0.1 μg/ml boiled FasL plus 0.5 μg/ml Enhancer (gray line), or isotype control mAbs (dotted gray line). Results are representative of four independent experiments. (B) Comparison of B7.2 (CD86) upregulation by LPS (left) or FasL plus Enhancer (right) in mouse D1 cells. (C) Treatment of DCs (D1 cells, left; mBM-DCs, right) by anti-CD95 antibody (Jo-2) with concentrations ranging between 0.01 and 10 μg/ml or an isotype control (thin line). Results are representative of three independent experiments.

Figure 3

Figure 3

Fas engagement triggers the phenotypical maturation of human and mouse DCs. (A) Surface expression of cellular receptors and intracellular expression of DC-LAMP in human CD34+–derived DCs treated with 0.1 μg/ml FasL plus 0.5 μg/ml Enhancer for 24 h (black line) versus 0.1 μg/ml boiled FasL plus 0.5 μg/ml Enhancer (gray line), or isotype control mAbs (dotted gray line). Results are representative of four independent experiments. (B) Comparison of B7.2 (CD86) upregulation by LPS (left) or FasL plus Enhancer (right) in mouse D1 cells. (C) Treatment of DCs (D1 cells, left; mBM-DCs, right) by anti-CD95 antibody (Jo-2) with concentrations ranging between 0.01 and 10 μg/ml or an isotype control (thin line). Results are representative of three independent experiments.

Figure 2

Figure 2

Fas does not induce apoptotic cell death of immature or mature cells. (A) Flow cytometry of human DCs treated with FasL (right) or with boiled FasL (left) stained with 7AAD (see Materials and Methods). (B) Mouse DCs were either left untreated or treated for 20 h with LPS only, with LPS first and then with anti-Fas Jo-2 antibody for an additional 6 h, or with anti-Fas Jo-2 antibody alone at the indicated concentrations. Cells single positive for annexin V–FITC are considered early apoptotic and are plotted in the graph after subtracting the percentage of untreated cells. (C) Western blot for the expression of FLIPs in cell extracts of human DCs either untreated (NT) or treated with TNF-α, with boiled FasL (B.FasL), or with FasL (FasL) for 24 h. (D) Quantitative measurements of relative FLIP expression of C (based on four independent experiments, ±SEM) as performed using the ONE D-Scan program.

Figure 4

Figure 4

Fas-matured D1 cells produce large amounts of TNF-α and IL-1β. Cytokine production was measured by ELISA after 20 h incubation with anti-Fas antibody at the concentrations indicated (α-Fas 20 h); 20 h with 10 μg/ml LPS and then 6 h with anti-Fas (LPS+α-Fas); 26 h with anti-Fas alone (α-Fas 26 h); 26 h with isotype control for Jo-2 antibody alone (Isotype 26 h); 26 h with LPS alone (LPS); or left untreated (NT). Results are representative of three independent experiments.

Figure 5

Figure 5

Fas-treated fresh mBM-DCs preactivated with LPS produce large amounts of IL-1β. Cytokine production was measured by ELISA after 20 h of treatment with 10 μg/ml LPS and then 6 h with anti-Fas at the indicated concentrations (LPS+α-Fas 6 h); 20 h with 10 μg/ml LPS and then 6 h with isotype control, at the indicated concentrations (LPS+Isot. 6h); 6 h with anti-Fas (α-Fas 6h); 26 h with LPS alone (LPS); or left untreated (NT). Results are representative of three independent experiments.

Figure 6

Figure 6

IL-1β and IFN-γ release is partly inhibited by Fas-comp during cognate DC–T cell interaction. (A) DO11.10 Tg T cell proliferative response to the OVA 327–339 peptide in the presence (♦) or absence of 0.5 μg/ml Fas-comp (▪). T cells were incubated with or without D8 cells, and T cell proliferation was measured by [3H]thymidine incorporation. (B) IL-2 level in culture supernatants from the experiment in A at 5 μg/ml of peptide with Fas-comp (Fas-comp, gray bars) or without Fas-comp (ctrl, white bars) (0.5 μg/ml). (C) IL-1β was greatly reduced in the presence of Fas-comp at 48 h. (D) IFN-γ secretion was reduced in the presence of Fas-comp at 24 h, and little cytokine production was observed in the absence of DCs (CD4+T). (E) IL-12 was almost undetectable (<5 pg/ml) at all analyzed time points, both with and without Fas-comp. Results are representative of three independent experiments.

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