Prostaglandin E2 and tumor necrosis factor alpha cooperate to activate human dendritic cells: synergistic activation of interleukin 12 production

Abstract

Interleukin (IL)-12 is a proinflammatory cytokine that contributes to innate resistance and to the development of antigen-specific T cell responses. Among other effects, prostaglandin E2 (PGE2) inhibits the production of IL-12 by macrophages activated with lipopolysaccharide (LPS). Here we investigated the effects of PGE2 on human dendritic cells (DCs) which develop in the presence of GM-CSF and IL-4. We demonstrate that in the absence of LPS, PGE2 dose dependently stimulated the production of IL-12 by DCs. Although PGE2 alone stimulated the production of low amounts of IL-12 only, it synergized with tumor necrosis factor (TNF)-alpha to induce high levels of IL-12 production by DCs. Addition of TNF-alpha in the absence of PGE2 had no effect on IL-12 production. Conversely, in the presence of LPS, PGE2 inhibited IL-12 production by DCs in a dose-dependent manner. The combination of PGE2 and TNF-alpha efficiently silenced mannose receptor-mediated endocytosis in DCs and readily induced neo-expression of the CD83 antigen. In addition, the expression of various surface antigens such as major histocompatibility complex class I and II, adhesion, as well as costimulatory molecules was upregulated by this treatment. The effects of PGE2 on IL-12 synthesis and CD83 expression could be mimicked by dibutyryl-cAMP and forskolin, indicating that they were due to the intracellular elevation of cAMP levels. DC treated with PGE2 and TNF-alpha were most potent in stimulating allogeneic T cell proliferation. Our data demonstrate that PGE2 contributes to the maturation of human DCs and that PGE2 can be a potent enhancer of IL-12 production by human DCs.

Figure 1

Stimulatory effects of PGE2 on IL-12 production by human DCs. Day-5 DCs were incubated with graded doses of PGE2 either alone (A) or in the presence of a constant dose of LPS (B) or TNF-α (C). Conversely, DCs were also incubated with graded doses of TNF-α in the presence of a constant dose of PGE2 (D). After 48 h of culture, supernatants were harvested and IL-12 levels were determined using a specific ELISA. Dose-dependent responses from two out of four independent experiments are shown.

Figure 2

Regulatory effects of PGE2 on CD83 expression and Ag uptake. (A) Day-5 DCs were incubated with PGE2 (1 μM), TNF-α (1,000 U/ml), LPS (10 ng/ml), or PGE2 plus TNF-α. After 48 h, cells were harvested and CD83 expression was measured by flow cytometry. The isotype control (IgG2b) is also presented (dotted lines). (B) Day-5 DCs were incubated with PGE2 (1 μM), TNF-α (1,000 U/ml), or PGE2 plus TNF-α. After 24 h, cells were harvested and incubated with FITC-DX for 30 min at 37°C (controls at 0°C, dotted lines), washed, and analyzed by flow cytometry.

Figure 3

Phenotypic changes of DCs cultured with PGE2 and TNF-α. Day-5 DCs were recultured in the absence (dotted lines) or presence of PGE2 (1 μM) plus TNF-α (1,000 U/ml) (bold lines) for 48 h and analyzed by flow cytometry for the surface expression of the indicated Ag using the antibodies listed in Materials and Methods.

Figure 4

Mimicry of PGE2 effects on IL-12 synthesis and CD83 expression by nonphysiologic modulators of cAMP. Day-5 DCs were incubated with db-cAMP (100 μM) or forskolin (10 μM) either alone or in combination with TNF-α (1,000 U/ml). After 48 h, supernatants were analyzed for the presence of IL-12 using a specific ELISA (A) and the cells were analyzed for CD83 expression by flow cytometry (B). Data are given as mean ± SEM of four independent experiments.

Figure 5

T cell stimulatory capacity of DCs cultured with PGE2. Day-5 DCs were recultured in the absence or presence of PGE2 (10 μM), TNF-α (1,000 U/ml), or PGE2 plus TNF-α. After 48 h the cells were washed, irradiated, and DCs were used as stimulators of allogeneic T cell proliferation. Proliferation was monitored by measuring [methyl-³H]thymidine uptake on day 5 of coculture. Each value represents the mean cpm of triplicate cultures with SD.