Selective induction of tumor necrosis receptor factor 6/decoy receptor 3 release by bacterial antigens in human monocytes and myeloid dendritic cells

Abstract

Tumor necrosis factor (TNF) receptor 6/decoy receptor 3 (TR6/DcR3) is an antiapoptosis soluble receptor of the TNF family produced by tumor cells. In this study, TR6 expression in human immune cells was investigated. TR6 mRNA and protein were detectable in selected antigen-presenting cells. Monocytes and myeloid-derived dendritic cells (MDC) released the protein exclusively following stimulation of Toll-like receptor 2 (TLR2) and TLR4 by gram-positive and gram-negative bacterial antigens. Plasmacytoid dendritic cells, activated by bacterial antigens via TLR9 or by viral infection, did not produce the protein. Similarly, activated T cells did not release TR6. The release of TR6 by MDC was dependent on the activation of p42/p44 mitogen-activated protein kinases, Src-like protein tyrosine kinases, and phosphatidylinositol 3-kinase, signaling pathways important for MDC maturation and survival. In agreement with the in vitro data, TR6 levels in serum were significantly elevated in patients with bacterial infections. Overall, these data suggest a novel role for TR6 in immune responses to bacteria.

FIG. 1.

TR6 mRNA expression in resting or stimulated immune cells. B cells were treated for 18 to 20 h with LPS (5 μg/ml) or S. aureus Cowan I (SAC) (10⁻⁵ dilution), T cells were treated with PHA (5 μg/ml) or immobilized anti-CD3 (1 μg/ml) and anti-CD28 (10 μg/ml) MAb, NK cells were treated with IL-2 (100 U/ml) and IL-12 (5 ng/ml), and monocytes (Mon.) were treated with LPS (100 ng/ml). TR6 mRNA expression was evaluated by quantitative RT-PCR. Results for one of three donors are shown.

FIG. 2.

Bacterial antigens induce TR6 mRNA expression in monocytes and MDC. Quantitative RT-PCR was conducted with RNA from cells stimulated for 18 to 20 h with bacterial antigens recognized by TLR2 (LTA, prepared from B. subtilis and used at 1 μg/ml), TLR4 (LPS, 100 ng/ml), or TLR9 (CpG-ODN 2006, 1 μg/ml). Cells were also stimulated with IFN-γ (100 U/ml), TNF-α (50 ng/ml), CD40L (3 μg/ml), and IFN-α (100 ng/ml), singly or in combination. Results for two of four donors are shown for each cell type.

FIG. 3.

Release of TR6 by immune cell types. Protein release was evaluated for monocytes, MDC, PDC, and T cells stimulated for 24 h with IFN-γ (100 U/ml); LPS (100 ng/ml); LTA derived from B. subtilis (LTA 1), S. aureus (LTA 2), or S. faecalis (LTA 3) (all used at 1 μg/ml); CpG-ODN 2006 (1 μg/ml); 5 × 10⁴ PFU of encephalomyocarditis virus (EMCV); immobilized anti-CD3 MAb (1 μg/ml); and PHA (5 μg/ml) in the presence of IL-2 (100 U/ml). Cell-free supernatants were collected and assayed for the presence of soluble TR6 receptor by ELISA. Results obtained for one of four donors are shown.

FIG. 4.

Elevated levels of TR6 in the sera of patients with bacterial infections. Peripheral blood was obtained from patients (n = 27) with various infectious diseases admitted to the Georgetown University Hospital. Control samples were drawn from healthy blood donors (n = 36). Levels of TR6 in serum (nanograms per milliliter) were measured by ELISA. The horizontal bars indicate the average for each group: 0.38 ± 0.06 ng/ml for healthy donors and 3.46 ± 0.42 ng/ml for infected donors (mean ± standard error of the mean). The P value (<0.0001) was determined by the Mann-Whitney U test.

FIG. 5.

Effects of signal transduction inhibitors on TR6 and TNF-α release from LPS- or LTA-stimulated MDC. The cells were incubated with LPS or LTA (from B. subtilis) in the absence or presence of 50 μM PD98059 (p42/p44 MAPK inhibitor), 1.25 μM SB203580 (p38 MAPK inhibitor), 1.25 μM herbimycin A (tyrosine kinase inhibitor), or 0.05 μM wortmannin (PI3K inhibitor). The release of TR6 (black bars) and TNF-α (hatched bars) in the culture supernatants was analyzed by ELISA. Data shown represent mean values ± standard deviations for independent experiments conducted with cells of three donors.